Reducing intron retention

ABSTRACT

Disclosed herein are methods, compositions, polynucleic acid polymers, assays, and kits for inducing processing of a partially processed mRNA transcript to remove a retained intron to produce a fully processed mRNA transcript that encodes a full-length functional form of a protein. Also described herein are methods and compositions for treating a disease or condition characterized by impaired production of a full-length functional form of a protein or for treating a disease or condition characterized by a defective splicing in a subject.

CROSS-REFERENCE

This application is a divisional of U.S. application Ser. No.15/148,303, filed May 6, 2016, a continuation of U.S. application Ser.No. 14/741,071, filed Jun. 16, 2015, which claims the benefit of UKPatent Application No: 1410693.4, filed Jun. 16, 2014, each of which areincorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 24, 2015, isnamed 47991-704-201-seqlist_ST25.txt and is 17.8 kilobytes in size.

BACKGROUND

Alternative splicing can be a frequent phenomenon in the humantranscriptome. Intron retention is one example of alternative splicingin which a partially processed mRNA retains retention of at least oneintron after undergoing partial splicing. In some instances, thepresence of a retained intron in a partially processed mRNA can preventor reduce translation of a functional protein.

SUMMARY

This invention relates to a method of reducing or preventing intronretention in a transcript (e.g., a partially processed mRNA transcript)and treatment or prevention of diseases related to inadvertent intronretention.

In some aspects, the invention discloses a method of prevention ortreatment of a disease in a subject comprising reducing the incidence ofintron retention in gene transcripts, wherein the disease is induced bydefective protein expression caused by the intron retention in the genetranscripts.

In some aspects, the invention discloses a method of modulating intronsplicing in a cell, comprising hybridizing a polynucleic acid polymer toa region of pre-mRNA, wherein the region comprises or consists of SEQ IDNO: 46, or a region having at least 95% identity to SEQ ID NO: 46.

In some aspects, the invention discloses a method of modulating intronsplicing in a cell, comprising hybridizing a polynucleic acid polymer toa region of pre-mRNA, wherein the region comprises or consists of SEQ IDNO: 46, or a region having at least 95% identity to SEQ ID NO: 46.

In some aspects, the invention discloses a method of modulating intronsplicing in a cell, comprising hybridizing a polynucleic acid polymer toa region of pre-mRNA, wherein the region comprises or consists of SEQ IDNO: 3, or a region having at least 95% identity to SEQ ID NO: 3.

In some aspects, the invention discloses a method of modulating intronsplicing in a cell, comprising hybridizing a polynucleic acid polymer toa region of pre-mRNA, wherein the region comprises or consists of asequence complementary to a sequence having at least 95% identity to anyof the group of sequences comprising SEQ ID NOs: 47 to 434; orcombinations thereof.

In some aspects, the invention discloses a polynucleic acid polymerwhich is antisense to at least part of a region of polynucleic acidpolymer comprising or consisting of SEQ ID NO: 46, or a region ofpolynucleic acid polymer comprising or consisting of a sequence havingat least 95% sequence identity to SEQ ID NO: 46.

In some aspects, the invention discloses a polynucleic acid polymerwhich is antisense to at least part of a region of polynucleic acidpolymer comprising or consisting of SEQ ID NO: 3, or a region ofpolynucleic acid polymer comprising or consisting of a sequence havingat least 95% sequence identity to SEQ ID NO: 3.

In some aspects, the invention discloses a polynucleic acid polymerwhich is antisense to at least part of a region of polynucleic acidpolymer, wherein the region comprises or consists of a sequencecomplementary to any of the group of sequences comprising SEQ ID NOs: 47to 434; or combinations thereof; or optionally a region of polynucleicacid polymer comprising or consisting of a sequence having at least 95%sequence identity to SEQ ID NOs: 47 to 434.

In some aspects, the invention discloses a polynucleic acid polymercomprising or consisting of a nucleic acid sequence having at least 95%identity to a sequence selected from any of the group comprising SEQ IDNO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 7; SEQ IDNO: 8; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 14; SEQID NO: 16; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22;SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO:29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 35; SEQ IDNO: 37; SEQ ID NO: 38; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 43; andSEQ ID NO: 44; or combinations thereof; and optionally wherein theuracil nucleotides are substituted with thymine nucleotides

In some aspects, the invention discloses a polynucleic acid polymercomprising or consisting of a nucleic acid sequence having at least 95%identity to a sequence selected from any of the group comprising SEQ IDNOs: 47 to 434; or combinations thereof; and optionally wherein theuracil nucleotides are substituted with thymine nucleotides.

In some aspects, the invention discloses a pharmaceutical compositionwhich comprises a polynucleic acid polymer that hybridizes to a targetsequence of a partially processed mRNA transcript which encodes aprotein and which comprises a retained intron, wherein the targetsequence is in between two G quadruplexes, wherein the polynucleic acidpolymer is capable of inducing splicing out of the retained intron fromthe partially processed mRNA transcript; and a pharmaceuticallyacceptable excipient and/or a delivery vehicle. In some instances, thepolynucleic acid polymer hybridizes to the retained intron of thepartially processed mRNA transcript.

In some aspects, the invention discloses a pharmaceutical compositionwhich comprises a polynucleic acid polymer that hybridizes to a targetsequence of a partially processed mRNA transcript which encodes aprotein and which comprises a retained intron, wherein the polynucleicacid polymer hybridizes to an intronic splicing regulatory element ofthe partially processed mRNA transcript, wherein the intronic splicingregulatory element comprises a first CCC motif, and wherein thepolynucleic acid polymer is capable of inducing splicing out of theretained intron from the partially processed mRNA transcript; and apharmaceutically acceptable excipient and/or a delivery vehicle.

In some instances, the intronic splicing regulatory element furthercomprises a second CCC motif. In some instances, the first CCC motif isabout 3 or more nucleotide bases from the second CCC motif. In someinstances, the polynucleic acid polymer hybridizes to an intronicsplicing regulatory element comprising a CCCAG or an AGGCC motif.

In some aspects, the invention discloses a pharmaceutical compositionwhich comprises a polynucleic acid polymer that hybridizes to a targetsequence of a partially processed mRNA transcript which encodes aprotein and which comprises a retained intron, wherein the polynucleicacid polymer hybridizes to a binding motif of the partially processedmRNA transcript, wherein the binding motif does not form a G quadruplex,and wherein the polynucleic acid polymer is capable of inducing splicingout of the retained intron from the partially processed mRNA transcript;and a pharmaceutically acceptable excipient and/or a delivery vehicle.

In some instances, the polynucleic acid polymer comprises a pyridinenucleotide at the 3′ terminal position and/or at the 5′ terminalposition. In some instances, the polynucleic acid polymer comprises twoconsecutive pyridine nucleotides at the 3′ terminal position and/or atthe 5′ terminal position.

In some aspects, the invention discloses a pharmaceutical compositionwhich comprises a polynucleic acid polymer that hybridizes to a targetsequence of a partially processed mRNA transcript which encodes aprotein and which comprises a retained intron, wherein the polynucleicacid polymer hybridizes to a binding motif of the partially processedmRNA transcript, and wherein the binding motif forms a hairpinstructure, wherein the polynucleic acid polymer is capable of inducingsplicing out of the retained intron from the partially processed mRNAtranscript; and a pharmaceutically acceptable excipient and/or adelivery vehicle.

In some instances, the polynucleic acid polymer is further capable ofdestabilizing the hairpin structure. In some instances, the deliveryvehicle comprises a cell penetrating peptide or a peptide-basednanoparticle. In some instances, the delivery vehicle is complexed withthe polynucleic acid polymer by ionic bonding.

In some instances, the polynucleic acid polymer is between about 10 andabout 50, about 10 and about 45, about 10 and about 40, about 10 andabout 30, about 10 and about 25, or about 10 and about 20 nucleotides inlength. In some instances, the sequence of the polynucleic acid polymeris at least 60%, 70%, 80%, 90%, or 95% or 100% complementary to a targetsequence of the partially processed mRNA transcript. In some instances,the sequence of the polynucleic acid polymer has 4 or less, 3 or less, 2or less, or 1 or less mismatches to a target sequence of the partiallyprocessed mRNA transcript.

In some instances, the polynucleic acid polymer is modified at thenucleoside moiety or at the phosphate moiety. In some instances, thepolynucleic acid polymer comprises one or more artificial nucleotidebases. In some instances, the one or more artificial nucleotide basescomprises 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl,2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), T-O— dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA) modified, locked nucleic acid (LNA),ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1′,5′-anhydrohexitol nucleic acids (HNA), morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, or 2′-fluoroN3-P5′-phosphoramidites. In some instances, the polynucleic acid polymeris modified at the 2′ hydroxyl group of the ribose moiety of thenucleoside moiety of the polynucleic acid polymer. In some instances,the modification at the 2′ hydroxyl group is by a 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O—dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) moiety. In some instances, a methyl group is added to the 2′hydroxyl group of the ribose moiety to generate a 2′-O-methyl ribosemoiety. In some instances, a methoxyethyl group is added to the 2′hydroxyl group of the ribose moiety to generate a 2′-O-methoxyethylribose moiety. In some instances, the modification at the 2′ hydroxylgroup is linked to the 4′ carbon by a methylene group. In someinstances, the ribose ring is substituted with a six member morpholinoring to generate a morpholino artificial nucleotide analogue. In someinstances, the phosphate backbone is substituted with anoligoglycine-like moiety to generate peptide nucleic acid (PNA). In someinstances, the phosphate backbone is modified by a thiol group or amethyl group. In some instances, the 5′ terminus, 3′ terminus, or acombination thereof is modified. In some instances, the modificationprotects the polynucleic acid polymer from endogenous nucleases in thesubject. In some instances, the modified polynucleic acid polymer doesnot induce or has a reduced ability to induce RNase H cleavage of RNA.In some instances, the polynucleic acid polymer is modified to increaseits stability. In some instances, the hybridization is specifichybridization.

In some instances, the polynucleic acid polymer hybridizes to an mRNAtranscript comprising at least 80%, 85%, 90%, or 95% or 100% sequenceidentity to at least 13 contiguous bases of SEQ ID NO: 46. In someinstances, the polynucleic acid polymer hybridizes to a mRNA transcriptcomprising at least 10 contiguous bases of SEQ ID NO: 46. In someinstances, the polynucleic acid polymer hybridizes to a mRNA transcriptcomprising at least 63%, 70%, 80%, 90%, or 95% sequence identity to asequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ IDNO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQID NO: 39, SEQ ID NO: 42, SEQ ID NO: 45, or combinations thereof. Insome instances, the polynucleic acid polymer hybridizes to a mRNAtranscript comprising at least 55%, 60%, 70%, 80%, 90%, or 95% sequenceidentity to SEQ ID NO: 3. In some instances, the polynucleic acidpolymer comprises at least 63%, 70%, 80%, 90%, or 95% or 100% sequenceidentity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32,SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO:40, SEQ ID NO: 41, SEQ ID NO: 43, and SEQ ID NO: 44, or combinationsthereof, and optionally wherein uracil nucleotides are substituted withthymine nucleotides. In some instances, the polynucleic acid polymercomprises at least 55%, 60%, 70%, 80%, 90%, or 95%, or comprises 100%sequence identity to a sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2, or combinations thereof, and optionallywherein uracil nucleotides are substituted with thymine nucleotides. Insome instances, the polynucleic acid polymer hybridizes to an mRNAtranscript comprising at least 80%, 85%, 90%, or 95% sequence identityto at least 13 contiguous bases of a sequence selected from the groupconsisting of SEQ ID NOs: 47-434, or combinations thereof.

In some instances, the polynucleic acid polymer is a synthesizedpolynucieic acid polymer.

In some instances, the pharmaceutical composition comprising thepolynucleic acid polymer is for intravenous or subcutaneousadministration.

In some aspects, the invention discloses a composition for use in thetreatment of a disease or condition in a patient in need thereof,comprising administering to the patient a pharmaceutical compositiondisclosed herein. In some instances, the disease or condition isassociated with an impaired production of a protein or is characterizedby a defective splicing. In some instances, the disease or condition isa hereditary disease. In some instances, a subject with the hereditarydisease has a genome that comprises a copy of a gene that comprises anexon that when properly transcribed into fully processed mRNA encodesthe full-length functional form of the protein. In some instances, asubject with the hereditary disease has a genome that comprises a copyof a gene that comprises a set of exons that when properly transcribedinto fully processed mRNA encodes the full-length functional form of theprotein. In some instances, a subject with the hereditary disease has agenome that comprises a defective copy of the gene, which is incapableof producing a full-length functional form of the protein. In someinstances, the disease or condition is diabetes. In some instances, thedisease or condition is cancer. In some instances, the composition foruse further comprises selecting a subject for treatment of a disease orcondition associated with an impaired production of a protein whichcomprises (a) determining if the subject has the disease or conditionassociated with an impaired production of the protein; and (b)administering to the subject a pharmaceutical composition describedabove if the subject has the disease or condition associated with animpaired production of the protein. In some instances, the compositionfor use further comprises selecting a subject for treatment of a diseaseor condition characterized by a defective splicing, which comprises (a)determining if the subject has the disease or condition characterized bythe defective splicing; and (b) administering to the subject apharmaceutical composition described above if the subject has thedisease or condition characterized by the defective splicing.

In some aspects, the invention discloses a method of treating a diseaseor condition characterized by impaired production of a full-lengthfunctional form of a protein in a subject in need thereof, comprising:(a) administering to the subject a pharmaceutical compositioncomprising: a therapeutic agent that induces an increase in splicing outof an intron in a partially processed mRNA transcript; and apharmaceutically acceptable excipient and/or a delivery vehicle; whereinthe subject has a pool of partially processed mRNA transcripts, whichare capable of encoding copies of the full-length functional form of theprotein and each of which comprise at least one retained intron thatinhibits translation of the partially processed mRNA transcripts; and(b) contacting a target cell of the subject with the therapeutic agentto induce a portion of the pool of the partially processed mRNAtranscripts to undergo splicing to remove the at least one retainedintron from each of the partially processed mRNA transcripts in theportion, to produce fully processed mRNA transcripts, wherein the fullyprocessed mRNA transcripts are translated to express copies of thefull-length functional form of the protein, which treat the disease orcondition.

In some instances, the therapeutic agent causes activation of one ormore splicing protein complexes in the cell to remove the at least oneretained intron from each of the partially processed mRNA transcripts inthe portion of the pool of the partially processed mRNA transcripts. Insome instances, the therapeutic agent inhibits a protein that regulatesintron splicing activity. In some instances, the therapeutic agentactivates a protein that regulates intron splicing activity. In someinstances, the therapeutic agent binds to a protein that regulatesintron splicing activity. In some instances, the therapeutic agent bindsto target polynucleotide sequence of the partially processed mRNAtranscripts. In some instances, the therapeutic agent is a polynucleicacid polymer. In some instances, the therapeutic agent is a smallmolecule.

In some instances, the pharmaceutical composition is the pharmaceuticalcomposition described herein.

In some instances, the impaired production of a full-length functionalform of the protein comprises sub-normal production of the full-lengthfunctional form of the protein. In some instances, the impairedproduction of a full-length functional form of the protein is due to anabsence of expression of the full-length functional form of the proteinor a level of expression of the full-length functional form of theprotein that is sufficiently low so as to cause the disease orcondition. In some instances, the impaired production of a full-lengthfunctional form of the protein comprises absence of production of theprotein or production of a defective form of the protein. In someinstances, the defective form of the protein is a truncated form of theprotein, a mis-folded form of the protein or a form of the protein withaberrant target binding. In some instances, treating the subject resultsin increased expression of the full-length functional form of theprotein.

In some aspects, the invention discloses a method of inducing processingof a partially processed mRNA transcript to remove a retained intron toproduce a fully processed mRNA transcript that encodes a full-lengthfunctional form of a protein, comprising: (a) hybridizing an isolatedpolynucleic acid polymer to the partially processed mRNA transcript,which is capable of encoding the full-length functional form of theprotein and which comprises at least one retained intron; (b) removingthe at least one retained intron from the partially processed mRNAtranscript to produce a fully processed mRNA transcript that encodes afull-length functional form of the protein; and (c) translating thefull-length functional form of the protein from the fully processed mRNAtranscript. In some instances, the method further comprisesadministering a pharmaceutical composition comprising the isolatedpolynucleic acid polymer to a subject in need thereof.

In some instances, the impaired production of the full-length functionalform of the protein is correlated to a disease or condition. In someinstances, the disease or condition is a hereditary disease. In someinstances, the subject with the hereditary disease has a genome thatcomprises a copy of a gene that comprises an exon that when properlytranscribed into fully processed mRNA encodes the full-length functionalform of the protein. In some instances, the subject with the hereditarydisease has a genome that comprises a copy of a gene that comprises aset of exons that when properly transcribed into fully processed mRNAencodes the full-length functional form of the protein. In someinstances, the subject with the hereditary disease has a genome thatcomprises a defective copy of the gene, which is incapable of producinga full-length functional form of the protein. In some instances, thedisease or condition is diabetes. In some instances, the disease orcondition is cancer.

In some instances, the polynucleic acid polymer is an anti-sensesequence. In some instances, the anti-sense sequence hybridizes to aretained intron of the partially processed mRNA transcript. In someinstances, the anti-sense sequence hybridizes to an intronic splicingregulatory element of the partially processed mRNA transcript. In someinstances, the intronic splicing regulatory element comprises a CCCmotif. In some instances, the anti-sense sequence hybridizes to abinding motif of the partially processed mRNA transcript wherein thebinding motif does not form a G quadruplex. In some instances, theanti-sense sequence hybridizes to a binding motif of the partiallyprocessed mRNA transcript wherein the binding motif is between two Gquadruplexes. In some instances, the anti-sense sequence hybridizes to abinding motif of the partially processed mRNA transcript wherein thebinding motif has a first CCC motif and a second CCC motif. In someinstances, the first CCC motif is about 3 or more nucleotide bases fromthe second CCC motif. In some instances, the sequence of the polynucleicacid polymer is at least 60%, 70%, 80%, 90%, or 95% or 100%complementary to a target sequence of the partially processed mRNAtranscript. In some instances, the sequence of the polynucleic acidpolymer has 4 or less, 3 or less, 2 or less, or 1 or less mismatches toa target sequence of the partially processed mRNA transcript. In someinstances, the polynucleic acid polymer is between about 10 and about50, about 10 and about 45, about 10 and about 40, about 10 and about 30,about 10 and about 25, or about 10 and about 20 nucleotides in length.

In some instances, the subject is a eukaryote. In some instances, thesubject is a eukaryote selected from a human, mouse, rat, non-humanprimate, or non-primate mammal.

In some aspects, described is a pharmaceutical composition whichcomprises a polynucleic acid polymer that hybridizes to a targetsequence of a partially processed mRNA transcript which encodes aprotein and which comprises a retained intron, wherein the polynucleicacid polymer induces splicing out of the retained intron from thepartially processed mRNA transcript; and a pharmaceutically acceptableexcipient and/or a delivery vehicle.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1. Location of SSOs in the human proinsulin gene. (A) Schematics ofthe INS reporter and its mRNA products. SSOs are shown as blackhorizontal bars below exons (numbered boxes) and below intron 1 (line);their sequences are in Table 2. Start and stop codons are denoted byarrowheads. Canonical (solid lines) and cryptic (dotted lines) splicingis shown above the primary transcript; designation of cryptic splicesites is in grey. SSOs targeting intron 1 segments de14-de17 are shownin the lower panel. (B) mRNA isoforms (numbered 1-6) generated by theINS reporter construct. Description of isoforms that do not produceproinsulin is labelled with *.

FIG. 2. SSO-induced inhibition of INS intron 1 retention. (A)Cotransfection of the INS reporter construct (IC D-F) with the indicatedSSOs into HEK293 cells. Spliced products described in FIG. 1B are shownto the right. Bars represent percentage of intron 1-containing isoformsrelative to natural transcripts (upper panel) or percentage of splicingto the cryptic 3′ splice site of intron 2 relative to the total (lowerpanel). Error bars denote SD; sc, scrambled control; SSO—, ‘no SSO’control. Final concentration of SSOs was 1, 3, 10 and 30 nM, except forSSO6 and SSO8 (10 and 30 nM). (B) SSO21-mediated promotion of intron 1splicing in clones lacking the cryptic 3ss of intron 2. RNA products areto the right. (C) A fold change in SSO21-induced intron 1 retention intranscripts containing and lacking the cryptic 3′ss of intron 2. Thefinal concentration of SSO21 was 30 nM in duplicate transfection.Designation of the reporter constructs is at the bottom.

FIG. 3. INS SSOs targeting cryptic 3′ splice sites. (A) Activation ofcryptic 3′ss of intron 2 (cr3′ss+126; FIG. 1A) by SSO6 and promotion ofexon 2 skipping by SSO8. Concentration of each SSO was 2, 10, 50 and 250nM. SSOs are shown at the top, spliced products to the right, reporterat the bottom. (B) A predicted stable hairpin between the authentic andcryptic 3′ss of INS intron 2. Bases targeted by SSO6 are denoted byasterisks and predicted splicing enhancer hexamers (listed to the right)are denoted by a dotted line. (C) SSO4 does not prevent activation ofcryptic 3′ss 81 base pairs downstream of its authentic counterpart(cr3′ss+81) in cells depleted of U2AF35 but induces exon skipping. Thefinal concentration of each SSO in COS7 cells was 5, 20 and 80 nM. Thefinal concentration of the siRNA duplex U2AF35ab (77) was 70 nM. Thereporter was the same as in panel A.

FIG. 4. Optimization of the intron retention target by antisensemicrowalk at a single-nucleotide resolution. (A) Location ofoligoribonucleotides. Microwalk SSOs and oligos used for CD/NMR arerepresented by horizontal black bars below and above the primarytranscript, respectively. Intron 1 sequences predicted to form RNAG-quadruplexes are highlighted in grey. Microwalk direction is shown bygrey arrows; winner oligos are highlighted in black. A box denotes asingle nucleotide polymorphism reported previously (20). (B) Intronretention levels of each microwalk SSO in two cell lines. Error barsdenote SDs obtained from two independent cotransfections with reporterIC D-F.

FIG. 5. Biophysical characterization of RNA secondary structureformation. (A) Far-UV CD spectrum at 25° C. for CD1 (19-mer) and CD2(20-mer) RNAs, revealing ellipticity maximum at 265 and 270 nm,respectively. (B) 1H NMR spectra of CD1 and CD2 recorded at 800 MHz and298 K showing characteristic groups of resonances from H-bonded G bases.(C) Sigmoidal CD melting curves for the two RNAs showing a transitionmid-point at 56.8±0.2° C. and 69.0±0.45° C., respectively. The twocurves have been displaced slightly from each other for clarity. (D) Theproposed parallel quadruplex structure with two stacked G-tetradsconnected by short loop sequences for CD1 (top panel). Predicted hairpinstructures for CD2 are shown at the bottom panel. G→C mutations areindicated by arrow.

FIG. 6. Conformational quadruplex/hairpin transitions involving theantisense target. (A) Schematic equilibrium between hairpin (black) andquadruplex (dark blue) structures proposed to form within the G-richmotif encompassing oligoribonucleotide CD3. CD4 contains a CC→UUmutation (highlighted by *). (B) The NMR spectrum in the 9-15 ppm regionreveals imino proton signals corresponding to hydrogen bonded bases. Thesignals between 10 and 12 ppm are characteristic of Hoogsteen hydrogenbonded Gs within a G-tetrad (Q box), while signals >12 ppm areindicative of Watson-Crick A-U and G-C base pairs within hairpinstructures (H box). In CD3, hairpin H1 is significantly populated, butmutations in CD4 destabilize H1 making H2 the major species, with bothin equilibrium with the quadruplex structure. (C) Mfold predictions oftwo possible hairpins, consistent with the NMR data. (D) Reduction ofintron retention upon destabilization of the hairpin structure by theCC→UU mutation. Error bars denote SD of a duplicate experiment withreporter IC D-C. De15, the IC D-C reporter lacking segment del5 (FIG.1A); M1, a reporter containing two substitutions (Table 1A) todestabilize both the G-quadruplex and the stem-loop.

FIG. 7. Identification of proteins that interact with pre-mRNAsencompassing the antisense target for intron retention. (A) Intronretention levels for wild type and mutated reporter constructs (IC D-C)following transient transfections into HEK293T cells. Mutations areshown in Table 1A. RNA products are to the right. The presence ofpredicted RNA quadruplexes, hairpins H1/H2 and the upstream anddownstream C4 run are indicated below the gel figure. Error bars denoteSDs obtained from two replicate experiments. (B) Intron retention levelsof tested RNAs correlate with their predicted stabilities across theantisense target. (C) Western blot analysis of a pull-down assay withantibodies indicated to the right. NE, nuclear extracts; B, beads-onlycontrol; AV3, control RNA oligo containing a cytosine run and a 3′ss AG(7). The sequence of CD5 RNA is shown in FIG. 4A.

FIG. 8. Splicing pattern of quadruplex-rich and -poor minigenes uponDHX36 depletion. (A) Schematics of reporter constructs. Predictedquadruplexes are denoted by black rectangles; their densities are shownin Table 1. Exons (boxes) are numbered; forward slash denotes shorteningof F9 intron 3 (24). The F9 and TSC2 minigenes contain branch pointsubstitutions c.253-25C and c.5069-18C, respectively, that impairsplicing (24). Cr5′ss-104; cryptic 5′ss 104 upstream of authentic 5′ssof intron 2. (B) Immunoblot with antibodies against DHX36. sc, scrambledsiRNA; c, untreated cells. Error bars are SDs of two transfectionexperiments. (C-E) Intron retention and exon skipping of the indicatedreporters. The final concentration of DHX36 siRNA was 50 nM. RNAproducts are shown schematically to the right. Error bars are SDs of twotransfection experiments.

DETAILED DESCRIPTION

Genes such as eukaryotic genes contain intervening sequences or intronsthat must be accurately removed from primary transcripts to createfunctional mRNAs capable of encoding proteins (1). This process modifiesmRNA composition in a highly dynamic manner, employing interdependentinteractions of five small nuclear RNAs (snRNAs) (e.g., U1, U2, U4, U5,and U6) and a large number of proteins with conserved but degeneratesequences in the pre-mRNA (2). In particular, introns can be defined bythe core splice site elements that comprises the 5′ splice site (5′ss)which can comprise a conserved GU motif, the 3′ splice site (3′ss) whichcan terminates with an invariant AG motif, the branchpoint sequencewhich can comprise a conserved adenine base, and the polypyrimidine (Py)tract. Splicing reaction can be initiated upon binding of the U1 snRNPto the conserved GU motif on the 5′ss, followed by binding of U2 snRNPat the conserved adenine base of the branchpoint, and finally U4, U5,and U6 snRNPs interactions near the 5′ and 3′ splice site. The complexformed by snRNPs and the respective intron can be referred to as aspliceosome. Additional splicing factors such as U2 small nuclear RNAauxiliary factor 1 (U2AF35), U2AF2 (U2AF65) and splicing factor 1 (SF1)can contribute to the spliceosome assembly and facilitate the splicingevent.

Intron splicing generally promotes mRNA accumulation and proteinexpression across species (3-5). This process can be altered by intronicmutations or variants that may also impair coupled gene expressionpathways, including transcription, mRNA export and translation. This isexemplified by introns in the 5′UTR where natural variants or mutationsmodifying intron retention alter the relative abundance of transcriptswith upstream open reading frames (uORFs) or other regulatory motifs anddramatically influence translation (6,7). Further, impaired proteintranslation due to a defective splicing such as intron retention has ledto development of diseases and/or progression of diseases such asgenetic disorders or conditions (e.g., hereditary diseases or cancer).However, successful sequence-specific strategies to normalize geneexpression in such situations have not been developed.

Splice-switching oligonucleotides (SSOs) are antisense reagents thatmodulate intron splicing by binding splice-site recognition orregulatory sequences and competing with cis- and trans-acting factorsfor their targets (8). They have been shown to restore aberrantsplicing, modify the relative expression of existing mRNAs or producenovel splice variants that are not normally expressed (8). Improvedstability of targeted SSO-RNA duplexes by a number of SSO modifications,such as 2′-O-methyl and 2′-O-methoxyethyl ribose, facilitated studiesexploring their therapeutic potential for a growing number of humandisease genes, including DMD in muscular dystrophy (9,10), SMN2 inspinal muscular atrophy (11), ATM in ataxia-telangiectasia (12) and BTKin X-linked agammaglobulinaemia (13). Although such approaches are closeto achieving their clinical potential for a restricted number ofdiseases (8), >300 Mendelian disorders resulting from mutation-inducedaberrant splicing (14) and a growing number of complex traits may beamenable to SSO-mediated correction of gene expression.

Etiology of type 1 diabetes has a strong genetic component conferred byhuman leukocyte antigens (HLA) and a number of modifying non-HLA loci(15). The strongest modifier was identified in the proinsulin gene (INS)region on chromosome 11 (termed IDDM2) (15). Further mapping of thisarea suggested that INS is the most likely IDDM2 target (16), consistentwith a critical role of this autoantigen in pathogenesis (17). Geneticrisk to this disease at IDDM2 has been attributed to differentialsteady-state RNA levels from predisposing and protective INS haplotypes,potentially involving a minisatellite DNA sequence upstream of this gene(18,19). However, systematic examination of naturally occurring INSpolymorphisms revealed haplotype-specific proinsulin expression levelsin reporter constructs devoid of the minisatellite sequence, resultingfrom two variants in intron 1 (7), termed IVS1+5ins4 (also known asrs3842740 or INS-69) and IVS1-6A/T (rs689, INS-27 or HphI+/−) (16,20).The former variant activates a cryptic 5′ splice site of intron 1whereas adenine (A) at the latter variant, which resides 6 nucleotidesupstream of the 3′ splice site (3′ss), promotes intron retention,expanding the relative abundance of transcripts with extended 5′UTR(21). As compared to thymine (T), the A allele at IVS1-6A/T decreasesaffinity to pyrimidine-binding proteins in vitro and renders the 3′ssmore dependent on the auxiliary factor of U2 small nuclearribonucleoprotein (U2AF) (7), a heterodimer required for U2 binding,spliceosome assembly and 3′ss selection (22). Intron 1-containingtranscripts are overrepresented in IVS1-6A-derived cDNA librariesprepared from insulin producing tissues (21), are exported from thenucleus (23), and contain a short, Homininae-specific uORF thatco-evolved with relaxation of the 3′ss of intron 1 in higher primates(7). The lower proinsulin expression conferred by the A allele may leadto suboptimal presentation of proinsulin peptides in the fetal thymusand inadequate negative selection of autoreactive T cells, culminatingin autoimmune destruction of insulin-producing β cells in the pancreas(7).

An aim of the invention is to induce processing of a partially processedmRNA transcript to remove a retained intron to produce a fully processedmRNA transcript that encodes a full-length functional form of a protein.An additional aim is to treat a disease or condition characterized byimpaired production of a protein and/or a disease or conditioncharacterized by a defective splicing in a subject in need thereof.

Another aim of the invention is to correct the low efficiency of INSintron 1 removal from the IVS1-6A-containing pre-mRNAs and reduce intronretention to the levels observed for the disease-protective T allele. Afurther aim of the invention is to provide new therapy approaches togenetic diseases including cancer that are characterized by (orassociated with) irregular or aberrant intron retention.

According to a first aspect of the invention, there is provided a methodof prevention or treatment of a disease in a subject comprisingcorrection of intron retention in mature gene transcripts, wherein thedisease is induced by defective protein expression caused by the intronretention in the gene transcripts.

Retained Intron

Retained intron is one of five types of alternative splicing that canalso include exon skipping, alternative 5′ splice site, alternative 3′splice site, and mutually exclusive exons. Exon skipping can occur whenan exon is skipped over or is spliced out of the processed mRNA and canbe the most common type of alternative splicing. Alternative 5′ss andalternative 3′ss can signify alternate splice sites such as crypticsplice sites or pseudo splice sites. Mutually exclusive exons can occurwhen only one of two exons is retained in the processed mRNA aftersplicing. Although intron retention can be less common than exonskipping, intron retention has been shown to occur more frequently thanpreviously realized. Indeed, a study of 21,106 human genes by the DeSouza group has shown that about 15% of the genes tested showed intronretention (see, Galante et al, “Detection and evaluation of intronretention events in the human transcriptome,” Bioinformatics 10:757-765(2004)). Further, a study by the Moore group has shown that about 35% ofhuman 5′-UTRs and about 16% of 3′-UTRs harbor introns (Bicknell et al.,“Introns in UTRs: Why we should stop ignoring them,” Bioessays34:1025-1034 (2012)). As such, intron retention can occur in a codingregion, a non-coding region, at the 5′ UTR, or at the 3′ UTR. In thecoding region, the retained intron can encode amino acids in frame, orcan be in misalignment which can generate truncated proteins ornon-functional proteins due to stop codon or frame shifts. Further,intron can be in between two exons, located at the 5′ UTR, or located atthe 3′ UTR.

Compared to a non-retained intron, a retained intron can becharacterized to have a shorter sequence length. The sequence length ofthe retained intron can be less than 5 kb, less than 4.5 kb, less than 4kb, less than 3.5 kb, less than 3 kb, less than 2.5 kb, less than 2 kb,less than 1.5 kb, less than 1 kb, less than 0.5 kb, less than 0.4 kb,less than 0.3 kb, less than 0.2 kb, or less than 0.1 kb.

Further, compared to a non-retained intron, a retained intron can becharacterized to have a G/C content of between about 40% to about 60%.The G/C content of the retained intron can be about 40%, 45%, 50%, 55%,or about 60%.

The retained intron can also be flanked by a weak 5′ splice site, a weak3′ splice site, or both. A weak splice site can refer to a splice sitethat may require a regulatory protein such as an intronic splicingenhancer for function.

Furthermore, a retained intron may comprise a lower presence of a GGGmotif relative to a non-retained intron. In some instances, the GGGmotif is an intronic splicing enhancer.

A partially processed mRNA transcript is a mRNA transcript that hasundergone partial splicing and comprises at least one retained intron.The at least one intron can be within the 5′ UTR, 3′ UTR, or at aninternal position in between two exons. The partially processed mRNAtranscript can be unable to be translated to produce a functional orfull-length protein.

A partially processed mRNA transcript can comprise an intron that ischaracterized by a short sequence length. A partially processed mRNAtranscript can comprise an intron that is characterized by a sequence ofless than 3 kb, less than 2.5 kb, less than 2 kb, less than 1.5 kb, lessthan 1 kb, less than 0.5 kb, less than 0.4 kb, less than 0.3 kb, lessthan 0.2 kb, or less than 0.1 kb.

A partially processed mRNA transcript can comprise an intron that ischaracterized to have a G/C content of between about 40% to about 60%. Apartially processed mRNA transcript can comprise an intron that ischaracterized to have a G/C content of about 40%, 45%, 50%, 55%, orabout 60%.

A partially processed mRNA transcript can comprise an intron that isflanked by a weak 5′ splice site, a weak 3′ splice site, or both.

A partially processed mRNA transcript can comprise an intron that maycomprise a lower presence of a GGG motif relative to a non-retainedintron.

In some cases, one or more introns are retained in a partially processedmRNA transcript. The partially processed mRNA can comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more retained introns.

A fully processed mRNA transcript is one that has undergone splicing toremove introns, such as retained introns and is capable of beingtranslated to produce a protein such as a full length functionalprotein. A partially processed mRNA transcript with one or more retainedintrons can be spliced so as to become a fully processed mRNA.

A full-length functional protein has the same length and function as thewild-type form of the protein. The full-length functional protein can bethe wild-type form of the protein. The full-length functional proteincan be an isoform of the wild type protein with the same length as thewild type protein. The full-length functional protein can comprise amutation such as a substitution of an amino acid for an alternativeamino acid residue with similar properties so that the phenotype (e.g.,the function) of the protein is not altered by the mutation.

Exemplary amino acids with similar properties can include amino acidswith electrically charged side chains: arginine, histidine, and lysinewhich are positively charged; or aspartic acid and glutamic acid whichare negatively charged; polar amino acid residues: serine, threonine,asparagine, glutamine, cysteine, and methionine; nonpolar amino acids:glycine, alanine, valine, leucine, isoleucine, and proline; and aromaticamino acids: phenylalanine, tyrosine, and tryptophan.

In some instances, the partially processed mRNA transcript leads toimpaired production of a protein. The impaired production of the proteincan be the impaired production of a full-length functional form of theprotein which can comprise sub-normal production of the full-lengthfunctional form of the protein. The impaired production of a full-lengthfunctional form of the protein can comprise production of a defectiveform of the protein. The defective form of the protein can be atruncated form of the protein, a mis-folded form of the protein, or aform of the protein that comprises an aberrant target binding site.

Polynucleic Acid Polymer

Polynucleic acid polymers described herein can be used to hybridize to apartially processed mRNA transcript to initiate removal of a retainedintron. In some instances, the polynucleic acid polymer is an antisensepolynucleic acid polymer. The antisense polynucleic acid polymer mayhybridize to a region of a partially processed mRNA transcript with ahigh G/C content, such as a region that comprises between about 40% toabout 60%. The antisense polynucleic acid polymer may comprise a highG/C content, such as between about 40% to about 60%. The antisensepolynucleic acid polymer may hybridize to a region of a partiallyprocessed mRNA transcript that is at, next to, or near a weak 5′ splicesite, or a weak 3′ splice site. The antisense polynucleic acid polymermay hybridize to a region of a partially processed mRNA transcript thatmay comprise a lower presence of a GGG motif.

The antisense sequence of the polynucleic acid polymer may hybridize toa retained intron of the partially processed mRNA transcript. Theantisense sequence may hybridize to an intronic splicing regulatoryelement of the partially processed mRNA transcript. The intronicsplicing regulatory element may include an intronic splicing enhancer oran intronic splicing silencer. The anti-sense sequence may hybridize toan intronic splicing silencer. The intronic splicing regulatory elementmay modulate splicing by affecting the early and/or intermediate stepsof spliceosomal assembly, such as when U1 and U2 snRNPs pair at splicesites across an exon during exon definition or during subsequenttransition to intron-spanning complex. Exemplary intronic splicingregulatory elements may include polypyrimidine-tract-binding proteinPTB, hnRNP L and hnRNP A1. Exemplary intronic splicing regulatoryelements may include binding sites for polypyrimidine-binding protein orPTB, U2AF65 and/or U2AF35, hnRNP A1 and hnRNP L.

The polynucleic acid polymer may hybridize at the 5′ splice site, 3′splice site, branchpoint, polypyrimidine tract, or an intron enhancer ofthe intron. The polynucleic acid polymer may also hybridize at adistance of about 30 bases away, 25 bases away, 20 bases away, 15 basesaway, 10 bases away, or 5 bases away from a 5′ splice site, 3′ splicesite, branchpoint, polypyrimidine tract or an intron enhancer to promoteor enhance splicing. Hybridization of the polynucleic acid polymer at ornear the splice sites may promote or enhance splicing by recruitingsplicing factors toward the splice sites, thereby initiating splicing.

A polynucleic acid polymer may hybridize at an intron silencer site(e.g., de novo intron silencer site), a cryptic intron splice site, or apseudo splice site. The intron silencer site can be recognized by asilencer which suppresses the splicing reaction. The cryptic intronsplice site can be created by a mutation, such as a substitution, adeletion, or an insertion. The cryptic intron splice site can be acryptic 5′ splice site or a cryptic 3′ splice site. The cryptic intronsplice site can be a cryptic 5′ splice site. The pseudo splice site canbe a weak splice site in which its activation can be resulted from amutation of the canonical splice site. The pseudo splice site can be apseudo 5′ splice site or a pseudo 3′ splice site. Hybridization at anintron silencer site may sterically block a silencer from binding andmay help from preventing disruption of the assembly and processing ofthe spliceosome. Hybridization at a cryptic intron splice site mayredirect interaction toward a canonical splice site such as a canonicalweak splice site.

A polynucleic acid polymer may hybridize to an internal region of theintron. The internal region of the intron may not encompass a splicesite, such as a 5′ splice site (e.g., canonical, cryptic, or pseudo 5′splice site), 3′ splice site (e.g., canonical, cryptic, or pseudo 3′splice site), an enhancer site or a silencer site. Hybridization of thepolynucleic acid polymer at an internal region may promote or enhancesplicing by recruiting splicing factors toward the splice sites, such asan intron enhancer site.

A polynucleic acid polymer may hybridize to an intronic splicingregulatory element of the partially processed mRNA transcript. Theintronic splicing regulatory element may comprise a CCC motif. Theanti-sense sequence may hybridize to a binding motif of the partiallyprocessed mRNA transcript wherein the binding motif does not form a Gquadruplex. The anti-sense sequence may hybridize to a binding motif ofthe partially processed mRNA transcript wherein the binding motif isbetween two G quadruplexes. The anti-sense sequence may also hybridizeto a binding motif of the partially processed mRNA transcript whereinthe binding motif has a first CCC motif and a second CCC motif. Thefirst CCC motif may be about 3 or more nucleotide bases from the secondCCC motif.

A polynucleic acid polymer may hybridize to a binding motif of thepartially processed mRNA transcript in which the binding motif forms ahairpin structure. The polynucleic acid polymer may further be capableof destabilizing the hairpin structure.

A polynucleic acid polymer may comprise a pyridine nucleotide at the 3′terminal position and/or at the 5′ terminal position. The polynucleicacid polymer may further comprise two consecutive pyridine nucleotidesat the 3′ terminal position and/or at the 5′ terminal position.

In some instances, a polynucleic acid polymer may be a splice-switchingoligonucleotide (SSO). The splice-switching oligonucleotide mayhybridize to a retained intron of the partially processed mRNAtranscript. The splice-switching oligonucleotide may hybridize to anintronic splicing regulatory element of the partially processed mRNAtranscript.

Splice-switching oligonucleotides (SSOs) have been widely used toinhibit exon usage but antisense strategies that promote removal ofentire introns to increase splicing-mediated gene expression have notbeen developed. Using a series of splicing reporters containing thehuman proinsulin gene, it has been shown that INS intron 1 retention bySSOs that bind transcripts derived from a human haplotype expressing lowlevels of proinsulin can be reduced. The SSO-assisted promotion of weakintron removal from the 5′UTR through competing noncanonical andcanonical RNA structures facilitates development of sequence-basedantisense strategies to enhance gene expression.

The term “correction of intron retention” is understood to thecorrection of irregular or aberrant intron retention. The correction maybe complete correction or partial correction. Correction may comprisereducing the incidence of intron retention. Correction may comprisereducing aberrant intron retention.

Reference to “defective” in the context of protein expression caused bythe intron retention in the gene transcripts, may comprise inadequate,defective, or aberrant protein expression.

Correction of intron retention may comprise administering a polynucleicacid polymer arranged to hybridize with the gene transcript. Correctionof intron retention may comprise administering a polynucleic acidpolymer arranged to hybridize with the gene transcript in order to alterhigher-order structures in the gene transcript. Correction of intronretention may comprise administering a polynucleic acid polymer arrangedto hybridize with the gene transcript in order to interfere with one ormore of conformational transitions of canonical (stem loops);noncanonical (G quadruplex) RNA structures; interactions withtrans-acting factors; and the rate of RNA-protein complex formation.Correction of intron retention may comprise administering a polynucleicacid polymer arranged to hybridize with the gene transcript in order tointerfere with, such as block, conformational transitions of canonical(stem loops) and/or noncanonical (G quadruplex) RNA structures. Thepolynucleic acid polymer may be antisense to at least part of a targetregion of the transcript comprising or consisting of intronic splicingregulatory elements. The polynucleic acid polymer may be antisense to atleast part of a target region of the transcript comprising or consistingof overlapped intronic splicing regulatory elements conserved inmammals. The polynucleic acid polymer may be antisense to at least partof a target region of the transcript comprising or consisting ofintronic segments containing short penta- to heptamer splicingregulatory motifs. Splicing regulatory motifs may comprise CCCAG orAGGCC. Splicing regulatory motifs may comprise any of the motifsprovided by Yeo G W, et al (2007). PLoS Genet 3:e85 and/or Voelker R B,& Berglund J A (2007). Genome Res 17:1023-1033, both documentsincorporated herein by reference. The target region may not comprise Cruns in order to avoid G-quadruplex formation. The target region may notbe proximal to both 5′ and 3′ splice sites, polypyrimidine tracts,branch sites and/or suprabranch regions.

The polynucleic acid polymer may provide binding platforms for splicingfactors which have been shown to influence INS intron 1 and exon 2splicing, including Tra2, SRSF3 or U2AF35, or other peptides, sense orantisense nucleic acids, small molecules, or other chemicals tofacilitate delivery of the polynucleic acid polymer and/or target thenucleic acid to a specific tissue, cell or a developmental stage. Suchantisense strategy may help reduce pervasive intron retention in cancercells, particularly those that contain somatic mutations of splicingfactor genes, as first shown for specific substitutions in the zincfinger domain of U2AF35 in myeloproliferative diseases (76). Thesemutations occur in many tumours and result in splicing defects that maycontribute to malignant growth. The invention may help control cellproliferation and reduce malignant growth in a significant fraction ofcancer patients that carry mutations in splicing factors involved in 3′splice site recognition, currently estimated at >15% (76).

The sequence of the polynucleic acid polymer may be at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% complementaryto a target sequence of the partially processed mRNA transcript. Thesequence of the polynucleic acid polymer may be 100% complementary to atarget sequence of the partially processed mRNA transcript.

The sequence of the polynucleic acid polymer may have 4 or lessmismatches to a target sequence of the partially processed mRNAtranscript. The sequence of the polynucleic acid polymer may have 3 orless mismatches to a target sequence of the partially processed mRNAtranscript. The sequence of the polynucleic acid polymer may have 2 orless mismatches to a target sequence of the partially processed mRNAtranscript. The sequence of the polynucleic acid polymer may have 1 orless mismatches to a target sequence of the partially processed mRNAtranscript.

The polynucleic acid polymer may specifically hybridize to a targetsequence of the partially processed mRNA transcript. The specificity maybe a 95%, 98%, 99%, 99.5% or 100% sequence complementarity of thepolynucleic acid polymer to a target sequence of the partially processedmRNA transcript. The hybridization may be under high stringenthybridization conditions.

The polynucleic acid polymer may be antisense to at least part of atarget region of the transcript comprising or consisting of SEQ ID NO:46. The polynucleic acid polymer may be antisense to at least part of atarget region of the transcript comprising or consisting of a sequencehaving at least 99% identity with SEQ ID NO: 46. The polynucleic acidpolymer may be antisense to at least part of a target region of thetranscript comprising or consisting of a sequence having at least 98%identity with SEQ ID NO: 46. The polynucleic acid polymer may beantisense to at least part of a target region of the transcriptcomprising or consisting of a sequence having at least 95% identity withSEQ ID NO: 46. The polynucleic acid polymer may be antisense to at leastpart of a target region of the transcript comprising or consisting of asequence having at least 90% identity with SEQ ID NO: 46. Thepolynucleic acid polymer may be antisense to at least part of a targetregion of the transcript comprising or consisting of a sequence havingat least 85% identity with SEQ ID NO: 46. The polynucleic acid polymermay be antisense to at least part of a target region of the transcriptcomprising or consisting of a sequence having at least 80% identity withSEQ ID NO: 46. The polynucleic acid polymer may be antisense to at leastpart of a target region of the transcript comprising or consisting of asequence having at least 75% identity with SEQ ID NO: 46. Thepolynucleic acid polymer may be antisense to at least part of a targetregion of the transcript comprising or consisting of a sequence havingat least 70% identity with SEQ ID NO: 46. The polynucleic acid polymermay be antisense to at least part of a target region of the transcriptcomprising or consisting of a sequence having at least 65% identity withSEQ ID NO: 46. The polynucleic acid polymer may be antisense to at leastpart of a target region of the transcript comprising or consisting of asequence having at least 60% identity with SEQ ID NO: 46. Thepolynucleic acid polymer may be antisense to at least part of a targetregion of the transcript comprising or consisting of a sequence havingat least 55% identity with SEQ ID NO: 46. The polynucleic acid polymermay be antisense to at least part of a target region of the transcriptcomprising or consisting of a sequence having at least 50% identity withSEQ ID NO: 46.

A polynucleic acid polymer may hybridize to an mRNA transcriptcomprising at least 80%, 85%, 90%, 95%, or 99% sequence identity to atleast 13 contiguous bases of SEQ ID NO: 46. The polynucleic acid polymermay hybridize to a mRNA transcript comprising 100% sequence identity toat least 13 contiguous bases of SEQ ID NO: 46.

A polynucleic acid polymer may be antisense to at least part of a targetregion of the transcript comprising or consisting of SEQ ID NO: 3. Thepolynucleic acid polymer may be antisense to at least part of a targetregion of the transcript comprising or consisting of a sequence havingat least 99% identity with SEQ ID NO: 3. The polynucleic acid polymermay be antisense to at least part of a target region of the transcriptcomprising or consisting of a sequence having at least 98% identity withSEQ ID NO: 3. The polynucleic acid polymer may be antisense to at leastpart of a target region of the transcript comprising or consisting of asequence having at least 95% identity with SEQ ID NO: 3. The polynucleicacid polymer may be antisense to at least part of a target region of thetranscript comprising or consisting of a sequence having at least 90%identity with SEQ ID NO: 3. The polynucleic acid polymer may beantisense to at least part of a target region of the transcriptcomprising or consisting of a sequence having at least 85% identity withSEQ ID NO: 3. The polynucleic acid polymer may be antisense to at leastpart of a target region of the transcript comprising or consisting of asequence having at least 80% identity with SEQ ID NO: 3. The polynucleicacid polymer may be antisense to at least part of a target region of thetranscript comprising or consisting of a sequence having at least 75%identity with SEQ ID NO: 3. The polynucleic acid polymer may beantisense to at least part of a target region of the transcriptcomprising or consisting of a sequence having at least 70% identity withSEQ ID NO: 3. The polynucleic acid polymer may be antisense to at leastpart of a target region of the transcript comprising or consisting of asequence having at least 65% identity with SEQ ID NO: 3. The polynucleicacid polymer may be antisense to at least part of a target region of thetranscript comprising or consisting of a sequence having at least 60%identity with SEQ ID NO: 3. The polynucleic acid polymer may beantisense to at least part of a target region of the transcriptcomprising or consisting of a sequence having at least 55% identity withSEQ ID NO: 3. The polynucleic acid polymer may be antisense to at leastpart of a target region of the transcript comprising or consisting of asequence having at least 50% identity with SEQ ID NO: 3.

Reference to antisense to at least part of a target region of thetranscript may comprise at least 5 consecutive nucleotides, or at least10 consecutive nucleotides. The polynucleic acid polymer may beantisense to at least 5 consecutive nucleotides of a target region ofthe transcript comprising or consisting of SEQ ID NO: 46. Thepolynucleic acid polymer may be antisense to at least 10 consecutivenucleotides of a target region of the transcript comprising orconsisting of SEQ ID NO: 46. The polynucleic acid polymer may beantisense to at least 15 consecutive nucleotides of a target region ofthe transcript comprising or consisting of SEQ ID NO: 46. Thepolynucleic acid polymer may be antisense to at least 20 consecutivenucleotides of a target region of the transcript comprising orconsisting of SEQ ID NO: 46.

A polynucleic acid polymer may hybridize to a mRNA transcript comprisingat least 10 contiguous bases of SEQ ID NO: 46. The polynucleic acidpolymer may hybridize to a mRNA transcript consisting of at least 10contiguous bases of SEQ ID NO: 46.

The polynucleic acid polymer may be antisense to a target region of thetranscript comprising or consisting of a sequence selected from any ofthe group comprising SEQ ID NO: 3; SEQ ID NO: 6; SEQ ID NO: 9; SEQ IDNO: 12; SEQ ID NO: 15; SEQ ID NO: 18; SEQ ID NO: 21; SEQ ID NO: 24; SEQID NO: 27; SEQ ID NO: 30; SEQ ID NO: 33; SEQ ID NO: 36; SEQ ID NO: 39;SEQ ID NO: 42; SEQ ID NO: 45; or combinations thereof.

A polynucleic acid polymer may be antisense to a target region of thetranscript comprising or consisting of a sequence having at least 99%sequence identity to a sequence selected from any of the groupcomprising SEQ ID NO: 3; SEQ ID NO: 6; SEQ ID NO: 9; SEQ ID NO: 12; SEQID NO: 15; SEQ ID NO: 18; SEQ ID NO: 21; SEQ ID NO: 24; SEQ ID NO: 27;SEQ ID NO: 30; SEQ ID NO: 33; SEQ ID NO: 36; SEQ ID NO: 39; SEQ ID NO:42; SEQ ID NO: 45; or combinations thereof. The polynucleic acid polymermay be antisense to a target region of the transcript comprising orconsisting of a sequence having at least 98% sequence identity to asequence selected from any of the group comprising SEQ ID NO: 3; SEQ IDNO: 6; SEQ ID NO: 9; SEQ ID NO: 12; SEQ ID NO: 15; SEQ ID NO: 18; SEQ IDNO: 21; SEQ ID NO: 24; SEQ ID NO: 27; SEQ ID NO: 30; SEQ ID NO: 33; SEQID NO: 36; SEQ ID NO: 39; SEQ ID NO: 42; SEQ ID NO: 45; or combinationsthereof. The polynucleic acid polymer may be antisense to a targetregion of the transcript comprising or consisting of a sequence havingat least 95% sequence identity to a sequence selected from any of thegroup comprising SEQ ID NO: 3; SEQ ID NO: 6; SEQ ID NO: 9; SEQ ID NO:12; SEQ ID NO: 15; SEQ ID NO: 18; SEQ ID NO: 21; SEQ ID NO: 24; SEQ IDNO: 27; SEQ ID NO: 30; SEQ ID NO: 33; SEQ ID NO: 36; SEQ ID NO: 39; SEQID NO: 42; SEQ ID NO: 45; or combinations thereof. The polynucleic acidpolymer may be antisense to a target region of the transcript comprisingor consisting of a sequence having at least 90% sequence identity to asequence selected from any of the group comprising SEQ ID NO: 3; SEQ IDNO: 6; SEQ ID NO: 9; SEQ ID NO: 12; SEQ ID NO: 15; SEQ ID NO: 18; SEQ IDNO: 21; SEQ ID NO: 24; SEQ ID NO: 27; SEQ ID NO: 30; SEQ ID NO: 33; SEQID NO: 36; SEQ ID NO: 39; SEQ ID NO: 42; SEQ ID NO: 45; or combinationsthereof. The polynucleic acid polymer may be antisense to a targetregion of the transcript comprising or consisting of a sequence havingat least 85% sequence identity to a sequence selected from any of thegroup comprising SEQ ID NO: 3; SEQ ID NO: 6; SEQ ID NO: 9; SEQ ID NO:12; SEQ ID NO: 15; SEQ ID NO: 18; SEQ ID NO: 21; SEQ ID NO: 24; SEQ IDNO: 27; SEQ ID NO: 30; SEQ ID NO: 33; SEQ ID NO: 36; SEQ ID NO: 39; SEQID NO: 42; SEQ ID NO: 45; or combinations thereof. The polynucleic acidpolymer may be antisense to a target region of the transcript comprisingor consisting of a sequence having at least 80% sequence identity to asequence selected from any of the group comprising SEQ ID NO: 3; SEQ IDNO: 6; SEQ ID NO: 9; SEQ ID NO: 12; SEQ ID NO: 15; SEQ ID NO: 18; SEQ IDNO: 21; SEQ ID NO: 24; SEQ ID NO: 27; SEQ ID NO: 30; SEQ ID NO: 33; SEQID NO: 36; SEQ ID NO: 39; SEQ ID NO: 42; SEQ ID NO: 45; or combinationsthereof. The polynucleic acid polymer may be antisense to a targetregion of the transcript comprising or consisting of a sequence havingat least 75% sequence identity to a sequence selected from any of thegroup comprising SEQ ID NO: 3; SEQ ID NO: 6; SEQ ID NO: 9; SEQ ID NO:12; SEQ ID NO: 15; SEQ ID NO: 18; SEQ ID NO: 21; SEQ ID NO: 24; SEQ IDNO: 27; SEQ ID NO: 30; SEQ ID NO: 33; SEQ ID NO: 36; SEQ ID NO: 39; SEQID NO: 42; SEQ ID NO: 45; or combinations thereof. The polynucleic acidpolymer may be antisense to a target region of the transcript comprisingor consisting of a sequence having at least 70% sequence identity to asequence selected from any of the group comprising SEQ ID NO: 3; SEQ IDNO: 6; SEQ ID NO: 9; SEQ ID NO: 12; SEQ ID NO: 15; SEQ ID NO: 18; SEQ IDNO: 21; SEQ ID NO: 24; SEQ ID NO: 27; SEQ ID NO: 30; SEQ ID NO: 33; SEQID NO: 36; SEQ ID NO: 39; SEQ ID NO: 42; SEQ ID NO: 45; or combinationsthereof. The polynucleic acid polymer may be antisense to a targetregion of the transcript comprising or consisting of a sequence havingat least 65% sequence identity to a sequence selected from any of thegroup comprising SEQ ID NO: 3; SEQ ID NO: 6; SEQ ID NO: 9; SEQ ID NO:12; SEQ ID NO: 15; SEQ ID NO: 18; SEQ ID NO: 21; SEQ ID NO: 24; SEQ IDNO: 27; SEQ ID NO: 30; SEQ ID NO: 33; SEQ ID NO: 36; SEQ ID NO: 39; SEQID NO: 42; SEQ ID NO: 45; or combinations thereof. The polynucleic acidpolymer may be antisense to a target region of the transcript comprisingor consisting of a sequence having at least 63% sequence identity to asequence selected from any of the group comprising SEQ ID NO: 3; SEQ IDNO: 6; SEQ ID NO: 9; SEQ ID NO: 12; SEQ ID NO: 15; SEQ ID NO: 18; SEQ IDNO: 21; SEQ ID NO: 24; SEQ ID NO: 27; SEQ ID NO: 30; SEQ ID NO: 33; SEQID NO: 36; SEQ ID NO: 39; SEQ ID NO: 42; SEQ ID NO: 45; or combinationsthereof. The polynucleic acid polymer may be antisense to a targetregion of the transcript comprising or consisting of a sequence havingat least 60% sequence identity to a sequence selected from any of thegroup comprising SEQ ID NO: 3; SEQ ID NO: 6; SEQ ID NO: 9; SEQ ID NO:12; SEQ ID NO: 15; SEQ ID NO: 18; SEQ ID NO: 21; SEQ ID NO: 24; SEQ IDNO: 27; SEQ ID NO: 30; SEQ ID NO: 33; SEQ ID NO: 36; SEQ ID NO: 39; SEQID NO: 42; SEQ ID NO: 45; or combinations thereof. The polynucleic acidpolymer may be antisense to a target region of the transcript comprisingor consisting of a sequence having at least 50% sequence identity to asequence selected from any of the group comprising SEQ ID NO: 3; SEQ IDNO: 6; SEQ ID NO: 9; SEQ ID NO: 12; SEQ ID NO: 15; SEQ ID NO: 18; SEQ IDNO: 21; SEQ ID NO: 24; SEQ ID NO: 27; SEQ ID NO: 30; SEQ ID NO: 33; SEQID NO: 36; SEQ ID NO: 39; SEQ ID NO: 42; SEQ ID NO: 45; or combinationsthereof.

The polynucleic acid polymer may be antisense to a target region of thetranscript comprising or consisting of a sequence selected from any ofthe group comprising SEQ ID NO: 3; SEQ ID NO: 6; SEQ ID NO: 9; and SEQID NO: 36; or combinations thereof. The polynucleic acid polymer may beantisense to a target region of the transcript comprising or consistingof a sequence having at least 99% identity to a sequence selected fromany of the group comprising SEQ ID NO: 3; SEQ ID NO: 6; SEQ ID NO: 9;and SEQ ID NO: 36; or combinations thereof. The polynucleic acid polymermay be antisense to a target region of the transcript comprising orconsisting of a sequence having at least 98% identity to a sequenceselected from any of the group comprising SEQ ID NO: 3; SEQ ID NO: 6;SEQ ID NO: 9; and SEQ ID NO: 36; or combinations thereof. Thepolynucleic acid polymer may be antisense to a target region of thetranscript comprising or consisting of a sequence having at least 95%identity to a sequence selected from any of the group comprising SEQ IDNO: 3; SEQ ID NO: 6; SEQ ID NO: 9; and SEQ ID NO: 36; or combinationsthereof.

The polynucleic acid polymer may be antisense to a target region of thetranscript comprising or consisting of SEQ ID NO: 3. The polynucleicacid polymer may be antisense to a target region of the transcriptcomprising or consisting of SEQ ID NO: 6. The polynucleic acid polymermay be antisense to a target region of the transcript comprising orconsisting of SEQ ID NO: 9. The polynucleic acid polymer may beantisense to a target region of the transcript comprising or consistingof SEQ ID NO: 36.

The polynucleic acid polymer may be antisense to a target region of thetranscript comprising or consisting of a sequence having at least 99%,at least 98%, at least 95%, at least 90%, at least 85%, at least 80%, atleast 75%, at least 70%, at least 65%, at least 63%, at least 60%, atleast 55%, or at least 50% identity to SEQ ID NO: 3. The polynucleicacid polymer may be antisense to a target region of the transcriptcomprising or consisting of a sequence having at least 99%, at least98%, at least 95%, at least 90%, at least 85%, at least 80%, at least75%, at least 70%, at least 65%, at least 63%, at least 60%, at least55%, or at least 50% identity to SEQ ID NO: 6. The polynucleic acidpolymer may be antisense to a target region of the transcript comprisingor consisting of a sequence having at least 99%, at least 98%, at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 63%, at least 60%, at least 55%, or at least50% identity to SEQ ID NO: 9. The polynucleic acid polymer may beantisense to a target region of the transcript comprising or consistingof a sequence having at least 99%, at least 98%, at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least65%, at least 63%, at least 60%, at least 55%, or at least 50% identityto SEQ ID NO: 36.

The polynucleic acid polymer may be antisense to a region of thetranscript comprising or consisting of a sequence complementary to anyof the group of sequences comprising SEQ ID NOs: 47 to 434; orcombinations thereof. The polynucleic acid polymer may be antisense to aregion of the transcript comprising or consisting of a sequencecomplementary to a sequence having at least 99% identity with any of thegroup of sequences comprising SEQ ID NOs: 47 to 434; or combinationsthereof. The polynucleic acid polymer may be antisense to a region ofthe transcript comprising or consisting of a sequence complementary to asequence having at least 98% identity with any of the group of sequencescomprising SEQ ID NOs: 47 to 434; or combinations thereof. Thepolynucleic acid polymer may be antisense to a region of the transcriptcomprising or consisting of a sequence complementary to a sequencehaving at least 95% identity with any of the group of sequencescomprising SEQ ID NOs: 47 to 434; or combinations thereof. Thepolynucleic acid polymer may be antisense to a region of the transcriptcomprising or consisting of a sequence complementary to a sequencehaving at least 90% identity with any of the group of sequencescomprising SEQ ID NOs: 47 to 434; or combinations thereof. Thepolynucleic acid polymer may be antisense to a region of the transcriptcomprising or consisting of a sequence complementary to a sequencehaving at least 85% identity with any of the group of sequencescomprising SEQ ID NOs: 47 to 434; or combinations thereof. Thepolynucleic acid polymer may be antisense to a region of the transcriptcomprising or consisting of a sequence complementary to a sequencehaving at least 80% identity with any of the group of sequencescomprising SEQ ID NOs: 47 to 434; or combinations thereof. Thepolynucleic acid polymer may be antisense to a region of the transcriptcomprising or consisting of a sequence complementary to a sequencehaving at least 75% identity with any of the group of sequencescomprising SEQ ID NOs: 47 to 434; or combinations thereof. Thepolynucleic acid polymer may be antisense to a region of the transcriptcomprising or consisting of a sequence complementary to a sequencehaving at least 70% identity with any of the group of sequencescomprising SEQ ID NOs: 47 to 434; or combinations thereof. Thepolynucleic acid polymer may be antisense to a region of the transcriptcomprising or consisting of a sequence complementary to a sequencehaving at least 65% identity with any of the group of sequencescomprising SEQ ID NOs: 47 to 434; or combinations thereof. Thepolynucleic acid polymer may be antisense to a region of the transcriptcomprising or consisting of a sequence complementary to a sequencehaving at least 60% identity with any of the group of sequencescomprising SEQ ID NOs: 47 to 434; or combinations thereof. Thepolynucleic acid polymer may be antisense to a region of the transcriptcomprising or consisting of a sequence complementary to a sequencehaving at least 55% identity with any of the group of sequencescomprising SEQ ID NOs: 47 to 434; or combinations thereof. Thepolynucleic acid polymer may be antisense to a region of the transcriptcomprising or consisting of a sequence complementary to a sequencehaving at least 50% identity with any of the group of sequencescomprising SEQ ID NOs: 47 to 434; or combinations thereof. The skilledperson will understand that uracil nucleotides may be substituted withthymine nucleotides (e.g. the DNA form of the RNA of such sequences).

The polynucleic acid polymer may comprise or consist of a polynucleicacid polymer sequence selected from any of the group comprising SEQ IDNO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 7; SEQ IDNO: 8; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 14; SEQID NO: 16; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22;SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO:29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 35; SEQ IDNO: 37; SEQ ID NO: 38; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 43; andSEQ ID NO: 44; or combinations thereof.

The polynucleic acid polymer may comprise or consist of a polynucleicacid polymer sequence having at least 99% identity to a sequenceselected from any of the group comprising SEQ ID NO: 1; SEQ ID NO: 2;SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10;SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO:17; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23; SEQ IDNO: 25; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 31; SEQID NO: 32; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 38;SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 43; and SEQ ID NO: 44; orcombinations thereof. The polynucleic acid polymer may comprise orconsist of a polynucleic acid polymer sequence having at least 98%identity to a sequence selected from any of the group comprising SEQ IDNO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 7; SEQ IDNO: 8; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 14; SEQID NO: 16; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22;SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO:29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 35; SEQ IDNO: 37; SEQ ID NO: 38; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 43; andSEQ ID NO: 44; or combinations thereof. The polynucleic acid polymer maycomprise or consist of a polynucleic acid polymer sequence having atleast 95% identity to a sequence selected from any of the groupcomprising SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 5; SEQID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 13; SEQID NO: 14; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 20;SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO:28; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 34; SEQ IDNO: 35; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 40; SEQ ID NO: 41; SEQID NO: 43; and SEQ ID NO: 44; or combinations thereof. The polynucleicacid polymer may comprise or consist of a polynucleic acid polymersequence having at least 90% identity to a sequence selected from any ofthe group comprising SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ IDNO: 5; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 11; SEQ IDNO: 13; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 19; SEQID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 26;SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO:34; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 40; SEQ IDNO: 41; SEQ ID NO: 43; and SEQ ID NO: 44; or combinations thereof. Thepolynucleic acid polymer may comprise or consist of a polynucleic acidpolymer sequence having at least 85% identity to a sequence selectedfrom any of the group comprising SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO:4; SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO:11; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 17; SEQ IDNO: 19; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 25; SEQID NO: 26; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 32;SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO:40; SEQ ID NO: 41; SEQ ID NO: 43; and SEQ ID NO: 44; or combinationsthereof. The polynucleic acid polymer may comprise or consist of apolynucleic acid polymer sequence having at least 80% identity to asequence selected from any of the group comprising SEQ ID NO: 1; SEQ IDNO: 2; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 8; SEQ IDNO: 10; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 16; SEQID NO: 17; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23;SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO:31; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 37; SEQ IDNO: 38; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 43; and SEQ ID NO: 44;or combinations thereof. The polynucleic acid polymer may comprise orconsist of a polynucleic acid polymer sequence having at least 75%identity to a sequence selected from any of the group comprising SEQ IDNO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 7; SEQ IDNO: 8; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 14; SEQID NO: 16; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22;SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO:29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 35; SEQ IDNO: 37; SEQ ID NO: 38; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 43; andSEQ ID NO: 44; or combinations thereof. The polynucleic acid polymer maycomprise or consist of a polynucleic acid polymer sequence having atleast 70% identity to a sequence selected from any of the groupcomprising SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 5; SEQID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 13; SEQID NO: 14; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 20;SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO:28; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 34; SEQ IDNO: 35; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 40; SEQ ID NO: 41; SEQID NO: 43; and SEQ ID NO: 44; or combinations thereof. The polynucleicacid polymer may comprise or consist of a polynucleic acid polymersequence having at least 65% identity to a sequence selected from any ofthe group comprising SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ IDNO: 5; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 11; SEQ IDNO: 13; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 19; SEQID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 26;SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO:34; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 40; SEQ IDNO: 41; SEQ ID NO: 43; and SEQ ID NO: 44; or combinations thereof. Thepolynucleic acid polymer may comprise or consist of a polynucleic acidpolymer sequence having at least 60% identity to a sequence selectedfrom any of the group comprising SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO:4; SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO:11; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 17; SEQ IDNO: 19; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 25; SEQID NO: 26; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 32;SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO:40; SEQ ID NO: 41; SEQ ID NO: 43; and SEQ ID NO: 44; or combinationsthereof. The polynucleic acid polymer may comprise or consist of apolynucleic acid polymer sequence having at least 55% identity to asequence selected from any of the group comprising SEQ ID NO: 1; SEQ IDNO: 2; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 8; SEQ IDNO: 10; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 16; SEQID NO: 17; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23;SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO:31; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 37; SEQ IDNO: 38; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 43; and SEQ ID NO: 44;or combinations thereof. The polynucleic acid polymer may comprise orconsist of a polynucleic acid polymer sequence having at least 50%identity to a sequence selected from any of the group comprising SEQ IDNO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 7; SEQ IDNO: 8; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 14; SEQID NO: 16; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22;SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO:29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 35; SEQ IDNO: 37; SEQ ID NO: 38; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 43; andSEQ ID NO: 44; or combinations thereof.

The polynucleic acid polymer may comprise or consist of a polynucleicacid polymer sequence selected from any of the group comprising SEQ IDNO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO 5; SEQ IN NO: 7; SEQ ID NO:8; SEQ ID NO: 34; and SEQ ID NO: 35; or combinations thereof. Thepolynucleic acid polymer may comprise or consist of a polynucleic acidpolymer sequence having at least 99% identity to a sequence selectedfrom any of the group comprising SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO:4; SEQ ID NO 5; SEQ IN NO: 7; SEQ ID NO: 8; SEQ ID NO: 34; and SEQ IDNO: 35; or combinations thereof. The polynucleic acid polymer maycomprise or consist of a polynucleic acid polymer sequence having atleast 98% identity to a sequence selected from any of the groupcomprising SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO 5; SEQ INNO: 7; SEQ ID NO: 8; SEQ ID NO: 34; and SEQ ID NO: 35; or combinationsthereof. The polynucleic acid polymer may comprise or consist of apolynucleic acid polymer sequence having at least 95% identity to asequence selected from any of the group comprising SEQ ID NO: 1; SEQ IDNO: 2; SEQ ID NO: 4; SEQ ID NO 5; SEQ IN NO: 7; SEQ ID NO: 8; SEQ ID NO:34; and SEQ ID NO: 35; or combinations thereof. The polynucleic acidpolymer may comprise or consist of a polynucleic acid polymer sequencehaving at least 90% identity to a sequence selected from any of thegroup comprising SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO 5;SEQ IN NO: 7; SEQ ID NO: 8; SEQ ID NO: 34; and SEQ ID NO: 35; orcombinations thereof. The polynucleic acid polymer may comprise orconsist of a polynucleic acid polymer sequence having at least 85%identity to a sequence selected from any of the group comprising SEQ IDNO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO 5; SEQ IN NO: 7; SEQ ID NO:8; SEQ ID NO: 34; and SEQ ID NO: 35; or combinations thereof. Thepolynucleic acid polymer may comprise or consist of a polynucleic acidpolymer sequence having at least 80% identity to a sequence selectedfrom any of the group comprising SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO:4; SEQ ID NO 5; SEQ IN NO: 7; SEQ ID NO: 8; SEQ ID NO: 34; and SEQ IDNO: 35; or combinations thereof. The polynucleic acid polymer maycomprise or consist of a polynucleic acid polymer sequence having atleast 75% identity to a sequence selected from any of the groupcomprising SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO 5; SEQ INNO: 7; SEQ ID NO: 8; SEQ ID NO: 34; and SEQ ID NO: 35; or combinationsthereof. The polynucleic acid polymer may comprise or consist of apolynucleic acid polymer sequence having at least 70% identity to asequence selected from any of the group comprising SEQ ID NO: 1; SEQ IDNO: 2; SEQ ID NO: 4; SEQ ID NO 5; SEQ IN NO: 7; SEQ ID NO: 8; SEQ ID NO:34; and SEQ ID NO: 35; or combinations thereof. The polynucleic acidpolymer may comprise or consist of a polynucleic acid polymer sequencehaving at least 65% identity to a sequence selected from any of thegroup comprising SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO 5;SEQ IN NO: 7; SEQ ID NO: 8; SEQ ID NO: 34; and SEQ ID NO: 35; orcombinations thereof. The polynucleic acid polymer may comprise orconsist of a polynucleic acid polymer sequence having at least 60%identity to a sequence selected from any of the group comprising SEQ IDNO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO 5; SEQ IN NO: 7; SEQ ID NO:8; SEQ ID NO: 34; and SEQ ID NO: 35; or combinations thereof. Thepolynucleic acid polymer may comprise or consist of a polynucleic acidpolymer sequence having at least 55% identity to a sequence selectedfrom any of the group comprising SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO:4; SEQ ID NO 5; SEQ IN NO: 7; SEQ ID NO: 8; SEQ ID NO: 34; and SEQ IDNO: 35; or combinations thereof. The polynucleic acid polymer maycomprise or consist of a polynucleic acid polymer sequence having atleast 50% identity to a sequence selected from any of the groupcomprising SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO 5; SEQ INNO: 7; SEQ ID NO: 8; SEQ ID NO: 34; and SEQ ID NO: 35; or combinationsthereof.

The polynucleic acid polymer may comprise or consist of SEQ ID NO: 1 orSEQ ID NO: 2. The polynucleic acid polymer may comprise or consist ofSEQ ID NO: 3 or SEQ ID NO: 4. The polynucleic acid polymer may compriseor consist of SEQ ID NO: 7 or SEQ ID NO: 8. The polynucleic acid polymermay comprise or consist of SEQ ID NO: 34 or SEQ ID NO: 35.

The polynucleic acid polymer may comprise or consist of a sequencehaving at least 99% identity to SEQ ID NO: 1 or SEQ ID NO: 2. Thepolynucleic acid polymer may comprise or consist of a sequence having atleast 98% identity to SEQ ID NO: 1 or SEQ ID NO: 2. The polynucleic acidpolymer may comprise or consist of a sequence having at least 95%identity to SEQ ID NO: 1 or SEQ ID NO: 2. The polynucleic acid polymermay comprise or consist of a sequence having at least 90% identity toSEQ ID NO: 1 or SEQ ID NO: 2. The polynucleic acid polymer may compriseor consist of a sequence having at least 85% identity to SEQ ID NO: 1 orSEQ ID NO: 2. The polynucleic acid polymer may comprise or consist of asequence having at least 80% identity to SEQ ID NO: 1 or SEQ ID NO: 2.The polynucleic acid polymer may comprise or consist of a sequencehaving at least 75% identity to SEQ ID NO: 1 or SEQ ID NO: 2. Thepolynucleic acid polymer may comprise or consist of a sequence having atleast 70% identity to SEQ ID NO: 1 or SEQ ID NO: 2. The polynucleic acidpolymer may comprise or consist of a sequence having at least 65%identity to SEQ ID NO: 1 or SEQ ID NO: 2. The polynucleic acid polymermay comprise or consist of a sequence having at least 60% identity toSEQ ID NO: 1 or SEQ ID NO: 2.

The polynucleic acid polymer may comprise or consist of a sequencehaving at least 99% identity to SEQ ID NO: 3 or SEQ ID NO: 4. Thepolynucleic acid polymer may comprise or consist of a sequence having atleast 98% identity to SEQ ID NO: 3 or SEQ ID NO: 4. The polynucleic acidpolymer may comprise or consist of a sequence having at least 95%identity to SEQ ID NO: 3 or SEQ ID NO: 4. The polynucleic acid polymermay comprise or consist of a sequence having at least 90% identity toSEQ ID NO: 3 or SEQ ID NO: 4. The polynucleic acid polymer may compriseor consist of a sequence having at least 85% identity to SEQ ID NO: 3 orSEQ ID NO: 4. The polynucleic acid polymer may comprise or consist of asequence having at least 80% identity to SEQ ID NO: 3 or SEQ ID NO: 4.The polynucleic acid polymer may comprise or consist of a sequencehaving at least 75% identity to SEQ ID NO: 3 or SEQ ID NO: 4. Thepolynucleic acid polymer may comprise or consist of a sequence having atleast 70% identity to SEQ ID NO: 3 or SEQ ID NO: 4. The polynucleic acidpolymer may comprise or consist of a sequence having at least 65%identity to SEQ ID NO: 3 or SEQ ID NO: 4. The polynucleic acid polymermay comprise or consist of a sequence having at least 60% identity toSEQ ID NO: 3 or SEQ ID NO: 4.

The polynucleic acid polymer may comprise or consist of a sequencehaving at least 99% identity to SEQ ID NO: 7 or SEQ ID NO: 8. Thepolynucleic acid polymer may comprise or consist of a sequence having atleast 98% identity to SEQ ID NO: 7 or SEQ ID NO: 8. The polynucleic acidpolymer may comprise or consist of a sequence having at least 95%identity to SEQ ID NO: 7 or SEQ ID NO: 8. The polynucleic acid polymermay comprise or consist of a sequence having at least 90% identity toSEQ ID NO: 7 or SEQ ID NO: 8. The polynucleic acid polymer may compriseor consist of a sequence having at least 85% identity to SEQ ID NO: 7 orSEQ ID NO: 8. The polynucleic acid polymer may comprise or consist of asequence having at least 80% identity to SEQ ID NO: 7 or SEQ ID NO: 8.The polynucleic acid polymer may comprise or consist of a sequencehaving at least 75% identity to SEQ ID NO: 7 or SEQ ID NO: 8. Thepolynucleic acid polymer may comprise or consist of a sequence having atleast 70% identity to SEQ ID NO: 7 or SEQ ID NO: 8. The polynucleic acidpolymer may comprise or consist of a sequence having at least 65%identity to SEQ ID NO: 7 or SEQ ID NO: 8. The polynucleic acid polymermay comprise or consist of a sequence having at least 60% identity toSEQ ID NO: 7 or SEQ ID NO: 8.

The polynucleic acid polymer may comprise or consist of a sequencehaving at least 99% identity to SEQ ID NO: 34 or SEQ ID NO: 35. Thepolynucleic acid polymer may comprise or consist of a sequence having atleast 98% identity to SEQ ID NO: 34 or SEQ ID NO: 35. The polynucleicacid polymer may comprise or consist of a sequence having at least 95%identity to SEQ ID NO: 34 or SEQ ID NO: 35. The polynucleic acid polymermay comprise or consist of a sequence having at least 90% identity toSEQ ID NO: 34 or SEQ ID NO: 35. The polynucleic acid polymer maycomprise or consist of a sequence having at least 85% identity to SEQ IDNO: 34 or SEQ ID NO: 35. The polynucleic acid polymer may comprise orconsist of a sequence having at least 80% identity to SEQ ID NO: 34 orSEQ ID NO: 35. The polynucleic acid polymer may comprise or consist of asequence having at least 75% identity to SEQ ID NO: 34 or SEQ ID NO: 35.The polynucleic acid polymer may comprise or consist of a sequencehaving at least 70% identity to SEQ ID NO: 34 or SEQ ID NO: 35. Thepolynucleic acid polymer may comprise or consist of a sequence having atleast 65% identity to SEQ ID NO: 34 or SEQ ID NO: 35. The polynucleicacid polymer may comprise or consist of a sequence having at least 60%identity to SEQ ID NO: 34 or SEQ ID NO: 35.

The polynucleic acid polymer may comprise or consist of a polynucleicacid polymer sequence selected from any of the group comprising SEQ IDNOs: 47 to 434; or combinations thereof. The polynucleic acid polymermay comprise or consist of a polynucleic acid polymer sequence selectedfrom any of the group comprising SEQ ID NOs: 47 to 434; or combinationsthereof, wherein uracil nucleotides are substituted with thyminenucleotides (e.g. the DNA form of the RNA of such sequences).

The polynucleic acid polymer may comprise or consist of a polynucleicacid polymer sequence having at least 99% identity with a sequenceselected from any of the group comprising SEQ ID NOs: 47 to 434; orcombinations thereof. The polynucleic acid polymer may comprise orconsist of a polynucleic acid polymer sequence having at least 99%identity with a sequence selected from any of the group comprising SEQID NOs: 47 to 434; or combinations thereof, wherein uracil nucleotidesare substituted with thymine nucleotides (e.g. the DNA form of the RNAof such sequences). The polynucleic acid polymer may comprise or consistof a polynucleic acid polymer sequence having at least 98% identity witha sequence selected from any of the group comprising SEQ ID NOs: 47 to434; or combinations thereof. The polynucleic acid polymer may compriseor consist of a polynucleic acid polymer sequence having at least 98%identity with a sequence selected from any of the group comprising SEQID NOs: 47 to 434; or combinations thereof, wherein uracil nucleotidesare substituted with thymine nucleotides (e.g. the DNA form of the RNAof such sequences). The polynucleic acid polymer may comprise or consistof a polynucleic acid polymer sequence having at least 95% identity witha sequence selected from any of the group comprising SEQ ID NOs: 47 to434; or combinations thereof. The polynucleic acid polymer may compriseor consist of a polynucleic acid polymer sequence having at least 95%identity with a sequence selected from any of the group comprising SEQID NOs: 47 to 434; or combinations thereof, wherein uracil nucleotidesare substituted with thymine nucleotides (e.g. the DNA form of the RNAof such sequences). The polynucleic acid polymer may comprise or consistof a polynucleic acid polymer sequence having at least 90% identity witha sequence selected from any of the group comprising SEQ ID NOs: 47 to434; or combinations thereof, wherein uracil nucleotides are substitutedwith thymine nucleotides (e.g. the DNA form of the RNA of suchsequences). The polynucleic acid polymer may comprise or consist of apolynucleic acid polymer sequence having at least 85% identity with asequence selected from any of the group comprising SEQ ID NOs: 47 to434; or combinations thereof, wherein uracil nucleotides are substitutedwith thymine nucleotides (e.g. the DNA form of the RNA of suchsequences). The polynucleic acid polymer may comprise or consist of apolynucleic acid polymer sequence having at least 80% identity with asequence selected from any of the group comprising SEQ ID NOs: 47 to434; or combinations thereof, wherein uracil nucleotides are substitutedwith thymine nucleotides (e.g. the DNA form of the RNA of suchsequences). The polynucleic acid polymer may comprise or consist of apolynucleic acid polymer sequence having at least 75% identity with asequence selected from any of the group comprising SEQ ID NOs: 47 to434; or combinations thereof, wherein uracil nucleotides are substitutedwith thymine nucleotides (e.g. the DNA form of the RNA of suchsequences). The polynucleic acid polymer may comprise or consist of apolynucleic acid polymer sequence having at least 70% identity with asequence selected from any of the group comprising SEQ ID NOs: 47 to434; or combinations thereof, wherein uracil nucleotides are substitutedwith thymine nucleotides (e.g. the DNA form of the RNA of suchsequences). The polynucleic acid polymer may comprise or consist of apolynucleic acid polymer sequence having at least 65% identity with asequence selected from any of the group comprising SEQ ID NOs: 47 to434; or combinations thereof, wherein uracil nucleotides are substitutedwith thymine nucleotides (e.g. the DNA form of the RNA of suchsequences). The polynucleic acid polymer may comprise or consist of apolynucleic acid polymer sequence having at least 60% identity with asequence selected from any of the group comprising SEQ ID NOs: 47 to434; or combinations thereof, wherein uracil nucleotides are substitutedwith thymine nucleotides (e.g. the DNA form of the RNA of suchsequences). The polynucleic acid polymer may comprise or consist of apolynucleic acid polymer sequence having at least 55% identity with asequence selected from any of the group comprising SEQ ID NOs: 47 to434; or combinations thereof, wherein uracil nucleotides are substitutedwith thymine nucleotides (e.g. the DNA form of the RNA of suchsequences). The polynucleic acid polymer may comprise or consist of apolynucleic acid polymer sequence having at least 50% identity with asequence selected from any of the group comprising SEQ ID NOs: 47 to434; or combinations thereof, wherein uracil nucleotides are substitutedwith thymine nucleotides (e.g. the DNA form of the RNA of suchsequences).

Advantageously, the invention identifies SSOs reducing the relativeabundance of intron transcripts (e.g., intron 1-retaining transcripts)and delineates the optimized antisense target at a single-nucleotideresolution.

A polynucleic acid polymer may comprise a splice-switchingoligonucleotide (SSO). The polynucleic acid polymer may be about 50nucleotides in length. The polynucleic acid polymer may be about 45nucleotides in length. The polynucleic acid polymer may be about 40nucleotides in length. The polynucleic acid polymer may be about 35nucleotides in length. The polynucleic acid polymer may be about 30nucleotides in length. The polynucleic acid polymer may be about 25nucleotides in length. The polynucleic acid polymer may be about 20nucleotides in length. The polynucleic acid polymer may be about 19nucleotides in length. The polynucleic acid polymer may be about 18nucleotides in length. The polynucleic acid polymer may be about 17nucleotides in length. The polynucleic acid polymer may be about 16nucleotides in length. The polynucleic acid polymer may be about 15nucleotides in length. The polynucleic acid polymer may be about 14nucleotides in length. The polynucleic acid polymer may be about 13nucleotides in length. The polynucleic acid polymer may be about 12nucleotides in length. The polynucleic acid polymer may be about 11nucleotides in length. The polynucleic acid polymer may be about 10nucleotides in length. The polynucleic acid polymer may be between about10 and about 50 nucleotides in length. The polynucleic acid polymer maybe between about 10 and about 45 nucleotides in length. The polynucleicacid polymer may be between about 10 and about 40 nucleotides in length.The polynucleic acid polymer may be between about 10 and about 35nucleotides in length. The polynucleic acid polymer may be between about10 and about 30 nucleotides in length. The polynucleic acid polymer maybe between about 10 and about 25 nucleotides in length. The polynucleicacid polymer may be between about 10 and about 20 nucleotides in length.The polynucleic acid polymer may be between about 15 and about 25nucleotides in length. The polynucleic acid polymer may be between about15 and about 30 nucleotides in length. The polynucleic acid polymer maybe between about 12 and about 30 nucleotides in length.

A polynucleic acid polymer, such as the SSOs, may comprise RNA or DNA.The polynucleic acid polymer, such as the SSOs, may comprise RNA. Thepolynucleic acid polymer, such as the SSOs, may comprise natural orsynthetic or artificial nucleotide analogues or bases, having equivalentcomplementation as DNA or RNA. The polynucleic acid polymer, such as theSSOs, may comprise combinations of DNA, RNA and/or nucleotide analogues.Nucleotide analogues may comprise PNA or LNA. In another embodiment, thenucleic acid, such as the SSOs, may comprise or consist of PMO.

In some instances, the synthetic or artificial nucleotide analogues orbases can comprise modifications at one or more of ribose moiety,phosphate moiety, nucleoside moiety, or a combination thereof.

Nucleotide analogues or artificial nucleotide base may comprise anucleic acid with a modification at a 2′ hydroxyl group of the ribosemoiety. The modification can be a 2′-O-methyl modification or a2′-O-methoxyethyl (2′-O-MOE) modification. The 2′-O-methyl modificationcan add a methyl group to the 2′ hydroxyl group of the ribose moietywhereas the 2′O-methoxyethyl modification can add a methoxyethyl groupto the 2′ hydroxyl group of the ribose moiety. Exemplary chemicalstructures of a 2′-O-methyl modification of an adenosine molecule and2′O-methoxyethyl modification of an uridine are illustrated below.

An additional modification at the 2′ hydroxyl group can include a2′-O-aminopropyl sugar conformation which can involve an extended aminegroup comprising a propyl linker that binds the amine group to the 2′oxygen. This modification can neutralize the phosphate derived overallnegative charge of the oligonucleotide molecule by introducing onepositive charge from the amine group per sugar and can thereby improvecellular uptake properties due to its zwitterionic properties. Anexemplary chemical structure of a 2′-O-aminopropyl nucleosidephosphoramidite is illustrated below.

Another modification at the 2′ hydroxyl group can include a locked orbridged ribose conformation (e.g., locked nucleic acid or LNA) where the4′ ribose position can also be involved. In this modification, theoxygen molecule bound at the 2′ carbon can be linked to the 4′ carbon bya methylene group, thus forming a 2′-C,4′-C-oxy-methylene-linkedbicyclic ribonucleotide monomer. Exemplary representations of thechemical structure of LNA are illustrated below. The representationshown to the left highlights the chemical connectivities of an LNAmonomer. The representation shown to the right highlights the locked3′-endo (³E) conformation of the furanose ring of an LNA monomer.

A further modification at the 2′ hydroxyl group may comprise ethylenenucleic acids (ENA) such as for example 2′-4′-ethylene-bridged nucleicacid, which locks the sugar conformation into a C₃′-endo sugar puckeringconformation. ENA are part of the bridged nucleic acids class ofmodified nucleic acids that also comprises LNA. Exemplary chemicalstructures of the ENA and bridged nucleic acids are illustrated below.

Still other modifications at the 2′ hydroxyl group can include 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O—dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA).

Nucleotide analogues may further comprise Morpholinos, peptide nucleicacids (PNAs), methylphosphonate nucleotides, thiolphosphonatenucleotides, 2′-fluoro N3-P5′-phosphoramidites, 1′, 5′-anhydrohexitolnucleic acids (HNAs), or a combination thereof. Morpholino orphosphorodiamidate morpholino oligo (PMO) comprises synthetic moleculeswhose structure mimics natural nucleic acid structure by deviates fromthe normal sugar and phosphate structures. Instead, the five memberribose ring can be substituted with a six member morpholino ringcontaining four carbons, one nitrogen and one oxygen. The ribosemonomers can be linked by a phosphordiamidate group instead of aphosphate group. These backbone alterations can remove all positive andnegative charges making morpholinos neutral molecules that can crosscellular membranes without the aid of cellular delivery agents such asthose used by charged oligonucleotides.

Peptide nucleic acid (PNA) does not contain sugar ring or phosphatelinkage. Instead, the bases can be attached and appropriately spaced byoligoglycine-like molecules, therefore, eliminating a backbone charge.

Modification of the phosphate backbone may also comprise methyl or thiolmodifications such as methylphosphonate nucleotide and. Exemplarythiolphosphonate nucleotide (left) and methylphosphonate nucleotide(right) are illustrated below.

Furthermore, exemplary 2′-fluoro N3-P5′-phosphoramidites is illustratedas:

And exemplary hexitol nucleic acid (or 1′, 5′-anhydrohexitol nucleicacids (HNA)) is illustrated as:

In addition to modification of the ribose moiety, phosphate backbone andthe nucleoside, the nucleotide analogues can also be modified by forexample at the 3′ or the 5′ terminus. For example, the 3′ terminus caninclude a 3′ cationic group, or by inverting the nucleoside at the3′-terminus with a 3′-3′ linkage. In another alternative, the3′-terminus can be blocked with an aminoalkyl group, e.g., a 3′C5-aminoalkyl dT. The 5-terminus can be blocked with an aminoalkylgroup, e.g., a 5′-O-alkylamino substituent. Other 5′ conjugates caninhibit 5′-3′ exonucleolytic cleavage. Other 3′ conjugates can inhibit3′-5′ exonucleolytic cleavage.

In some cases, one or more of the artificial nucleotide analoguesdescribed herein are resistant toward nucleases such as for exampleribonuclease such as RNase H, deoxyribunuclease such as DNase, orexonuclease such as 5′-3′ exonuclease and 3′-5′ exonuclease whencompared to natural polynucleic acid polymers. In some instances,artificial nucleotide analogues comprising 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O—dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, 2′-fluoroN3-P5′-phosphoramidites, or combinations thereof are resistant towardnucleases such as for example ribonuclease such as RNase H,deoxyribunuclease such as DNase, or exonuclease such as 5′-3′exonuclease and 3′-5′ exonuclease. 2′-O-methyl modified polynucleic acidpolymer may be nuclease resistance (e.g., RNase H, DNase, 5′-3′exonuclease or 3′-5′ exonuclease resistance). 2′O-methoxyethyl(2′-O-MOE) modified polynucleic acid polymer may be nuclease resistance(e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). 2′-O-aminopropyl modified polynucleic acid polymer may benuclease resistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistance). 2′-deoxy modified polynucleic acid polymer maybe nuclease resistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistance). T-deoxy-2′-fluoro modified polynucleic acidpolymer may be nuclease resistance (e.g., RNase H, DNase, 5′-3′exonuclease or 3′-5′ exonuclease resistance). 2′-O-aminopropyl (2′-O-AP)modified polynucleic acid polymer may be nuclease resistance (e.g.,RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance).2′-O-dimethylaminoethyl (2′-O-DMAOE) modified polynucleic acid polymermay be nuclease resistance (e.g., RNase H, DNase, 5′-3′ exonuclease or3′-5′ exonuclease resistance). 2′-O-dimethylaminopropyl (2′-O-DMAP)modified polynucleic acid polymer may be nuclease resistance (e.g.,RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance).T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE) modified polynucleic acidpolymer may be nuclease resistance (e.g., RNase H, DNase, 5′-3′exonuclease or 3′-5′ exonuclease resistance). 2′-O—N-methylacetamido(2′-O-NMA) modified polynucleic acid polymer may be nuclease resistance(e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). LNA modified polynucleic acid polymer may be nucleaseresistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). ENA modified polynucleic acid polymer may be nucleaseresistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). HNA modified polynucleic acid polymer may be nucleaseresistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). Morpholinos may be nuclease resistance (e.g., RNase H,DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance). PNA can beresistant to nucleases (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistance). Methylphosphonate nucleotides modifiedpolynucleic acid polymer may be nuclease resistance (e.g., RNase H,DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance).Thiolphosphonate nucleotides modified polynucleic acid polymer may benuclease resistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistance). Polynucleic acid polymer comprising 2′-fluoroN3-P5′-phosphoramidites may be nuclease resistance (e.g., RNase H,DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance).

In some instances, one or more of the artificial nucleotide analoguesdescribed herein have increased binding affinity toward their mRNAtarget relative to an equivalent natural polynucleic acid polymer. Theone or more of the artificial nucleotide analogues comprising2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O—dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, or 2′-fluoroN3-P5′-phosphoramidites can have increased binding affinity toward theirmRNA target relative to an equivalent natural polynucleic acid polymer.2′-O-methyl modified polynucleic acid polymer can have increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid polymer. 2′-O-methoxyethyl (2′-O-MOE) modifiedpolynucleic acid polymer can have increased binding affinity towardtheir mRNA target relative to an equivalent natural polynucleic acidpolymer. 2′-O-aminopropyl modified polynucleic acid polymer can haveincreased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid polymer. 2′-deoxy modifiedpolynucleic acid polymer can have increased binding affinity towardtheir mRNA target relative to an equivalent natural polynucleic acidpolymer. T-deoxy-2′-fluoro modified polynucleic acid polymer can haveincreased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid polymer. 2′-O-aminopropyl (2′-O-AP)modified polynucleic acid polymer can have increased binding affinitytoward their mRNA target relative to an equivalent natural polynucleicacid polymer. 2′-O-dimethylaminoethyl (2′-O-DMAOE) modified polynucleicacid polymer can have increased binding affinity toward their mRNAtarget relative to an equivalent natural polynucleic acid polymer.2′-O-dimethylaminopropyl (2′-O-DMAP) modified polynucleic acid polymercan have increased binding affinity toward their mRNA target relative toan equivalent natural polynucleic acid polymer.T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE) modified polynucleic acidpolymer can have increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid polymer.2′-O—N-methylacetamido (2′-O-NMA) modified polynucleic acid polymer canhave increased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid polymer. LNA modified polynucleicacid polymer can have increased binding affinity toward their mRNAtarget relative to an equivalent natural polynucleic acid polymer. ENAmodified polynucleic acid polymer can have increased binding affinitytoward their mRNA target relative to an equivalent natural polynucleicacid polymer. PNA modified polynucleic acid polymer can have increasedbinding affinity toward their mRNA target relative to an equivalentnatural polynucleic acid polymer. HNA modified polynucleic acid polymercan have increased binding affinity toward their mRNA target relative toan equivalent natural polynucleic acid polymer. Morpholino modifiedpolynucleic acid polymer can have increased binding affinity towardtheir mRNA target relative to an equivalent natural polynucleic acidpolymer. Methylphosphonate nucleotides modified polynucleic acid polymercan have increased binding affinity toward their mRNA target relative toan equivalent natural polynucleic acid polymer. Thiolphosphonatenucleotides modified polynucleic acid polymer can have increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid polymer. Polynucleic acid polymer comprising 2′-fluoroN3-P5′-phosphoramidites can have increased binding affinity toward theirmRNA target relative to an equivalent natural polynucleic acid polymer.The increased affinity can be illustrated with a lower Kd, a higher melttemperature (Tm), or a combination thereof.

In additional instances, a polynucleic acid polymer described herein maybe modified to increase its stability. In an embodiment where thepolynucleic acid polymer is RNA, the polynucleic acid polymer may bemodified to increase its stability. The polynucleic acid polymer may bemodified by one or more of the modifications described above to increaseits stability. The polynucleic acid polymer may be modified at the 2′hydroxyl position, such as by 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl(2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modification or by alocked or bridged ribose conformation (e.g., LNA or ENA). Thepolynucleic acid polymer may be modified by 2′-O-methyl and/or2′-O-methoxyethyl ribose. The polynucleic acid polymer may also includemorpholinos, PNAs, HNA, methylphosphonate nucleotides, thiolphosphonatenucleotides, or 2′-fluoro N3-P5′-phosphoramidites to increase itsstability. Suitable modifications to the RNA to increase stability fordelivery will be apparent to the skilled person.

A polynucleic acid polymer described herein can be constructed usingchemical synthesis and/or enzymatic ligation reactions using proceduresknown in the art. For example, a polynucleic acid polymer can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the polynucleic acid polymer and target nucleicacids. Exemplary methods can include those described in: U.S. Pat. No.5,142,047; U.S. Pat. No. 5,185,444; WO2009099942; or EP1579015.Additional exemplary methods can include those described in: Griffey etal., “2′-O-aminopropyl ribonucleotides: a zwitterionic modification thatenhances the exonuclease resistance and biological activity of antisenseoligonucleotides,” J. Med. Chem. 39(26):5100-5109 (1997)); Obika, et al.“Synthesis of 2′-O,4′-C-methyleneuridine and -cytidine. Novel bicyclicnucleosides having a fixed C3, -endo sugar puckering”. TetrahedronLetters 38 (50): 8735 (1997); Koizumi, M. “ENA oligonucleotides astherapeutics”. Current opinion in molecular therapeutics 8 (2): 144-149(2006); and Abramova et al., “Novel oligonucleotide analogues based onmorpholino nucleoside subunits-antisense technologies: new chemicalpossibilities,” Indian Journal of Chemistry 48B:1721-1726 (2009).Alternatively, the polynucleic acid polymer can be produced biologicallyusing an expression vector into which a polynucleic acid polymer hasbeen subcloned in an antisense orientation (i.e., RNA transcribed fromthe inserted polynucleic acid polymer will be of an antisenseorientation to a target polynucleic acid polymer of interest).

A polynucleic acid polymer may be bound to any nucleic acid molecule,such as another antisense molecule, a peptide, or other chemicals tofacilitate delivery of the polynucleic acid polymer and/or target thenucleic acid to a specific tissue, cell type, or cell developmentalstage. The polynucleic acid polymer may be bound to a protein or RNA.The protein tethered to the polynucleic acid polymer may comprise asplicing factor to enhance, inhibit or modulate splicing and intronremoval. RNA tethered to the polynucleic acid polymer may comprise anaptamer or any structure that enhance, inhibit or modulate splicing andintron removal. The polynucleic acid polymer may be isolated nucleicacid.

A polynucleic acid polymer may be conjugated to, or bound by, a deliveryvehicle suitable for delivering the polynucleic acid polymer to cells.The cells may be a specific cell type, or specific developmental stage.The delivery vehicle may be capable of site specific, tissue specific,cell specific or developmental stage-specific delivery. For example, thedelivery vehicle may be a cell specific viral particle, or componentthereof, alternatively, the delivery vehicle may be a cell specificantibody particle, or component thereof. The polynucleic acid polymermay be targeted for delivery to beta cells in the pancreas. Thepolynucleic acid polymer may be targeted for delivery to thymic cells.The polynucleic acid polymer may be targeted for delivery to malignantcells. The polynucleic acid polymer may be targeted for delivery topre-malignant cells (that are known to develop into overt malignantphenotypes within a foreseeable future, such as pre-leukaemias andmyelodysplastic syndromes or histopathologically defined precancerouslesions or conditions.

In one embodiment the polynucleic acid polymer may be bound to achemical molecule (e.g. non-peptide or nucleic acid based molecule),such as a drug. The drug may be a small molecule (e.g. having a MW ofless than 900 Da).

In one embodiment of the invention, the delivery vehicle may comprise acell penetrating peptide (CPP). For example, the polynucleic acidpolymer may be bound or complexed with a CPP. The skilled person willunderstand that any suitable CPP may be conjugated with the polynucleicacid polymer to aid delivery of the polynucleic acid polymer to and/orinto cells. Such CPPs may be any suitable CPP technology described byBoisguérin et al. (2015, Advanced Drug Delivery Reviews doi:10.1016/j.addr.2015.02.008), which is herein incorporated by reference.Suitable delivery vehicles for conjugation to the polynucleic acidpolymer are also described in Lochmann et al ((European Journal ofPharmaceutics and Biopharmaceutics 58 (2004) 237-251), which is hereinincorporated by reference).

The CPP may be an arginine and/or lysine rich peptide, for example,wherein the majority of residues in the peptide is either lysine orarginine. The CPP may comprise a poly-L-lysine (PLL). Alternatively, theCPP may comprise a poly-arginine. Suitable CPPs may be selected from thegroup comprising Penetratin; R6-Penetratin; Transportan;oligo-arginines; F-3; B-peptide; B-MSP; Pip peptides, such as Pip1,Pip2a, Pip2b, Pip5e, Pip5f, Pip5h, Pip5j; Pip5k, Pip5l, Pip5m, Pip5n,Pip5o, Pip6a, Pip6b, Pip6c, Pip6d, Pip6e, Pip6f, Pip6g, or Pip6h;peptide of sequence PKKKRKV; Penatratin; Lys₄; SPACE; Tat; Tat-DRBD(dsRNA-binding domain); (RXR)₄; (RFF)₃RXB; (KFF)₃K; R_(g)F₂; T-cellderived CPP; Pep-3; PEGpep-3; MPG-8; MPG-8-Chol; PepFect6; P5RHH; R₁₅;and Chol-R₉; or functional variants thereof (e.g. see Boisguérin et al.(2015, Advanced Drug Delivery Reviews doi: 10.1016/j.addr.2015.02.008).

In one embodiment, the CPP comprises or consists of a Pip peptide. ThePip peptide may be selected from the group comprising Pip1, Pip2a,Pip2b, Pip5e, Pip5f, Pip5h, Pip5j; Pip5k, Pip5l, Pip5m, Pip5n, Pip5o,Pip6a, Pip6b, Pip6c, Pip6d, Pip6e, Pip6f, Pip6g, and Pip6h.

In one embodiment of the invention, the delivery vehicle may comprise apeptide-based nanoparticle (PBN), wherein a plurality of CPPs (forexample one or more suitable CPPs discussed herein) form a complex withthe polynucleic acid polymer through charge interactions. Suchnanoparticles may be between about 50 nm and 250 nm in size. In oneembodiment the nanoparticles may be about 70-200 nm in size. In anotherembodiment the nanoparticles may be about 70-100 nm in size or 125-200nm in size.

In one embodiment, the polynucleic acid polymer may be complexed with adelivery vehicle, for example by ionic bonding. Alternatively, thepolynucleic acid polymer may be covalently bound to the deliveryvehicle. Conjugation/binding methods are described in Lochmann et al((European Journal of Pharmaceutics and Biopharmaceutics 58 (2004)237-251), which is herein incorporated by reference). For example, aconjugation method may comprise introducing a suitable tether containinga reactive group (e.g. —NH2 or —SH2) to the polynucleic acid polymer andto add the delivery vehicle, such as a peptide, post-synthetically as anactive intermediate, followed by carrying out the coupling reaction inaqueous medium. An alternative method may comprise carrying out theconjugation in a linear mode on a single solid-phase support.

The delivery vehicle and polynucleic acid polymer may be thiol and/ormaleimide linked, such as thiol-maleimide linked. The conjugation of thepolynucleic acid polymer and the delivery vehicle may be byclick-chemistry, such as reaction of azido or 2′-O-propyargyl functionalgroups and alkyne groups on the respective molecules to be conjugated.In one embodiment, the delivery vehicle and polynucleic acid polymer maybe linked by a thioether bridge. In another embodiment, the deliveryvehicle and polynucleic acid polymer may be linked by a disulphidebridge. The skilled person will readily identify suitable linking groupsor reactions for conjugation of polynucleic acid polymer and thedelivery vehicle, such as a peptide.

The gene transcript may encode pro-insulin. The gene transcript may betranscribed from INS gene. The gene transcripts may be derived from ahuman haplotype expressing low levels of proinsulin. The intron maycomprise INS intron 1.

The gene transcript may be transcribed from a gene or ORF selected fromany of the genes or ORFs comprising ABCD4; ABCF3; ACADVL; ALKBH6; AP1G2;APEX1; ARFRP1; ATHL1; ATP1A3; ATP5D; ATP13A1; BAX; BDH2; BRD2; C1orf63;C1orf630; Clorf631; Clorf124; C2orf49; C8orf82; C16orf59; CAPRIN2;CDCA7; CEP164; CEP170; CLCN7; CPNE1; CPSF3L; DCXR; DENND4B; DFFA;DIS3L2; DNAJB12; DPF1; DRG2; DSN1; EML3; EWSR1; EWSR10; FGFR4; FTSJ1;GBAP1; GMPPA; GMPR2; GNPTG; GORASP1; GPATCH4; HGS; HMG20B; IFFO1;ISYNA1; KRI1; LOC148413; LZTR1; MAN2C1; MAP4K2; MCOLN1; MDP1; MIB2;MITD1; MOK; MOV10; MRPL35; MTMR11; MUS81; NAPEPLD; NBEAL2; NDRG4;NDUFB10; NFATC4; NFKBIB; NIT1; NKTR; NPRL2; NSUN5P1; NUDT22; PAN2;PDDC1; PDLIM4; PHF1; PIK3CD; PITPNM1; PPIL2; PPP1R35; PPP4C; PQLC2;PRPF39; PSME2; PTPMT1; QARS; RAD52; RHOT2; RMND5B; RNF123; RPL10A;RPP21; RPS6KB2; RUSC1; SCRN2; SCYL1; SFR1; SGSM3; SIRT7; SLC25A3;SLC25A3; SLC30A7; SLC37A4; STK19; STX10; TCF25; TOMM40; TP53I3; TRIM41;TRPT1; TSTA3; TTC14; TTC140; TUBGCP6; U2AF1L4; UCK1; UNC45A; VAMP1;VAMP10; VARS; VPS28; WDR24; WDR90; WRAP53; YDJC; YIPF3; YIPF3; ZCCHC8;ZCCHC18; ZFAND1; ZNF131; ZNF300; ZNF317; ZNF692; ZNF711; ZNRD1; ZWINT;or combinations thereof.

The terms “polynucleic acid polymer” and “nucleic acid” can be usedinterchangeable and can refer to a polynucleic acid polymer that isbetween about 10 to about 50 nucleotides in length.

Diseases

The methods and compositions described herein can be used to treat adisease or condition characterized by an impaired production of aprotein. The methods and compositions described herein can also be usedto treat a disease or condition characterized by a defective splicing.The disease or condition can be a genetic disorder or condition. Thegenetic disorder or condition can be characterized by an impairedproduction of a protein. The genetic disorder or condition can also becharacterized by a defective splicing. The genetic disorder or conditioncan be a hereditary disorder, or a nonhereditary defect within one ormore locations within the genome. The genetic disorder can be ahereditary disease. The hereditary disease can be characterized by animpaired production of a protein. The hereditary disease can becharacterized by a defective splicing. A subject with a hereditarydisease can have a genome that can comprise a copy of a gene thatcomprises an exon that when properly transcribed into fully processedmRNA can encode the full-length functional for of the protein. A subjectwith a hereditary disease can have a genome that comprises a copy of agene that can comprise a copy of a gene that comprises a set of exonsthat when properly transcribed into fully processed mRNA can encode thefull-length functional form of the protein. A subject with a hereditarydisease can have a genome that can comprise a defective copy of thegene, which can be incapable of producing a full-length functional formof the protein.

The genetic disorder or condition can be a nonhereditary defect withinone or more locations within the genome. The nonhereditary defect can bea point mutation, a deletion, an insertion, or a frame shift. Thegenetic disorder or condition associated with the nonhereditary defectcan be characterized by an impaired production of a protein. The geneticdisorder or condition associated with the nonhereditary defect can becharacterized by a defective splicing. A subject with a nonhereditarydefect can have a genome that can comprise a copy of a gene thatcomprises an exon that when properly transcribed into fully processedmRNA can encode the full-length functional for of the protein. A subjectwith a nonhereditary defect can have a genome that comprises a copy of agene that can comprise a copy of a gene that comprises a set of exonsthat when properly transcribed into fully processed mRNA can encode thefull-length functional form of the protein. A subject with anonhereditary defect can have a genome that can comprise a defectivecopy of the gene, which can be incapable of producing a full-lengthfunctional form of the protein.

The genetic disorder or condition can be an autosomal dominant disorder,an autosomal recessive disorder, X-linked dominant disorder, X-linkedrecessive disorder, Y-linked disorder, mitochondrial disease, ormultifactorial or polygenic disorder. Sometimes, a hereditary diseasecan also be characterized as an autosomal dominant, autosomal recessive,X-linked dominant, X-linked recessive, Y-linked, mitochondrial, ormultifactorial or polygenic hereditary disease. Autosomal dominantdisorder, an autosomal recessive disorder, X-linked dominant disorder,X-linked recessive disorder, Y-linked disorder, mitochondrial disease,or multifactorial or polygenic disorder can be characterized by animpaired production of a protein. Autosomal dominant disorder, anautosomal recessive disorder, X-linked dominant disorder, X-linkedrecessive disorder, Y-linked disorder, mitochondrial disease, ormultifactorial or polygenic disorder can be characterized by a defectivesplicing. A subject with an autosomal dominant disorder, an autosomalrecessive disorder, X-linked dominant disorder, X-linked recessivedisorder, Y-linked disorder, mitochondrial disease, or multifactorial orpolygenic disorder can have a genome that can comprise a copy of a genethat comprises an exon that when properly transcribed into fullyprocessed mRNA can encode the full-length functional for of the protein.A subject with an autosomal dominant disorder, an autosomal recessivedisorder, X-linked dominant disorder, X-linked recessive disorder,Y-linked disorder, mitochondrial disease, or multifactorial or polygenicdisorder can have a genome that comprises a copy of a gene that cancomprise a copy of a gene that comprises a set of exons that whenproperly transcribed into fully processed mRNA can encode thefull-length functional form of the protein. A subject with an autosomaldominant disorder, an autosomal recessive disorder, X-linked dominantdisorder, X-linked recessive disorder, Y-linked disorder, mitochondrialdisease, or multifactorial or polygenic disorder can have a genome thatcan comprise a defective copy of the gene, which can be incapable ofproducing a full-length functional form of the protein.

Exemplary hereditary disease can include achondroplasia, hereditaryhemochromatosis, Down Syndrome, hereditary spherocytosis, Tay-SachsDisease, Usher syndrome, hereditary fructose intolerance, hemophilia,muscular dystrophy (e.g., Duchenne muscular dystrophy or DMD), polygenicdisorders, breast cancer, ovarian cancer, Parkinson's disease,Bardet-Biedl syndrome, Prader-Willi syndrome, diabetes, heart disease,arthritis, motor neuron disease, albinism, Cri-du-Chat syndrome, cysticfibrosis, fragile X syndrome, galactosemia, Huntington's disease,Jackson-Weiss syndrome, Klinefelter syndrome, Krabbe disease,Langer-Giedion syndrome, Lesch-Nyhan syndrome, Marfan syndrome, myotonicdystrophy, Nail-Patella syndrome, neurofibromatosis, Noonan syndrome,triple X syndrome, osteogenesis imperfecta, Patau syndrome,phenylketonuria, porphyria, retinoblastoma, Rett syndrome, sickle celldisease, Turner syndrome, Usher syndrome, Von Hippel-Lindau syndrome,Waardenburg syndrome, Wilson's disease, xeroderma pigmentosum, XXXXsyndrome, or YY syndrome.

A hereditary disease such as for example: achondroplasia, hereditaryhemochromatosis, Down Syndrome, hereditary spherocytosis, Tay-SachsDisease, Usher syndrome, hereditary fructose intolerance, hemophilia,muscular dystrophy (e.g., Duchenne muscular dystrophy or DMD), polygenicdisorders, breast cancer, ovarian cancer, Parkinson's disease,Bardet-Biedl syndrome, Prader-Willi syndrome, diabetes, heart disease,arthritis, motor neuron disease, albinism, Cri-du-Chat syndrome, cysticfibrosis, fragile X syndrome, galactosemia, Huntington's disease,Jackson-Weiss syndrome, Klinefelter syndrome, Krabbe disease,Langer-Giedion syndrome, Lesch-Nyhan syndrome, Marfan syndrome, myotonicdystrophy, Nail-Patella syndrome, neurofibromatosis, Noonan syndrome,triple X syndrome, osteogenesis imperfecta, Patau syndrome,phenylketonuria, porphyria, retinoblastoma, Rett syndrome, sickle celldisease, Turner syndrome, Usher syndrome, Von Hippel-Lindau syndrome,Waardenburg syndrome, Wilson's disease, xeroderma pigmentosum, XXXXsyndrome, or YY syndrome can be characterized by an impaired productionof a protein, or by a defective splicing. A hereditary disease such asfor example: achondroplasia, hereditary hemochromatosis, Down Syndrome,hereditary spherocytosis, Tay-Sachs Disease, Usher syndrome, hereditaryfructose intolerance, hemophilia, muscular dystrophy (e.g., Duchennemuscular dystrohy or DMD), polygenic disorders, breast cancer, ovariancancer, Parkinson's disease, Bardet-Biedl syndrome, Prader-Willisyndrome, diabetes, heart disease, arthritis, motor neuron disease,albinism, Cri-du-Chat syndrome, cystic fibrosis, fragile X syndrome,galactosemia, Huntington's disease, Jackson-Weiss syndrome, Klinefeltersyndrome, Krabbe disease, Langer-Giedion syndrome. Lesch-Nyhan syndrome,Marfan syndrome, myotonic dystrophy, Nail-Patella syndrome,neurofibromatosis, Noonan syndrome, triple X syndrome, osteogenesisimperfecta, Patau syndrome, phenylketonuria, porphyria, retinoblastoma,Rett syndrome, sickle cell disease, Turner syndrome, Usher syndrome, VonHippel-Lindau syndrome, Waardenburg syndrome, Wilson's disease,xeroderma pigmentosum, XXXX syndrome, or YY syndrome can comprise a copyof a gene that comprises an exon that when properly transcribed intofully processed mRNA can encode the full-length functional for of theprotein, can comprises a copy of a gene that can comprise a copy of agene that comprises a set of exons that when properly transcribed intofully processed mRNA can encode the full-length functional form of theprotein, or can comprise a defective copy of the gene, which can beincapable of producing a full-length functional form of the protein.

As described above, the genetic disorder or condition can be anautosomal dominant disorder, an autosomal recessive disorder, X-linkeddominant disorder, X-linked recessive disorder, Y-linked disorder,mitochondrial disease, or multifactorial or polygenic disorder. Thegenetic disorder or condition can be an autosomal dominant disorder, anautosomal recessive disorder, X-linked dominant disorder, X-linkedrecessive disorder, Y-linked disorder, mitochondrial disease, ormultifactorial or polygenic disorder can be a disorder characterized byan impaired production of a protein or by a defective splicing.

Exemplary autosomal dominant disorder can include Huntington's disease,neurofibromatosis type 1, neurofibromatosis type 2, Marfan syndrome,hereditary nonpolyposis colorectal cancer, hereditary multipleexostoses, Tuberous sclerosis, Von Willebrand disease, or acuteintermittent porphyria.

An autosomal dominant disorder such as Huntington's disease,neurofibromatosis type 1, neurofibromatosis type 2, Marfan syndrome,hereditary nonpolyposis colorectal cancer, hereditary multipleexostoses, Tuberous sclerosis, Von Willebrand disease, or acuteintermittent porphyria can be characterized by an impaired production ofa protein or by a defective splicing. An autosomal dominant disordersuch as Huntington's disease, neurofibromatosis type 1,neurofibromatosis type 2, Marfan syndrome, hereditary nonpolyposiscolorectal cancer, hereditary multiple exostoses, Tuberous sclerosis,Von Willebrand disease, or acute intermittent porphyria can comprise acopy of a gene that comprises an exon that when properly transcribedinto fully processed mRNA can encode the full-length functional for ofthe protein, can comprises a copy of a gene that can comprise a copy ofa gene that comprises a set of exons that when properly transcribed intofully processed mRNA can encode the full-length functional form of theprotein, or can comprise a defective copy of the gene, which can beincapable of producing a full-length functional form of the protein.

Exemplary autosomal recessive disorder can include albinism,Medium-chain acyl-CoA dehydrogenase deficiency, cystic fibrosis,sickle-cell disease, Tay-Sachs disease, Niemann-Pick disease, spinalmuscular atrophy, or Roberts syndrome.

An autosomal recessive disorder such as: albinism, Medium-chain acyl-CoAdehydrogenase deficiency, cystic fibrosis, sickle-cell disease,Tay-Sachs disease, Niemann-Pick disease, spinal muscular atrophy, orRoberts syndrome can be characterized by an impaired production of aprotein or by a defective splicing. An autosomal recessive disorder suchas: albinism, Medium-chain acyl-CoA dehydrogenase deficiency, cysticfibrosis, sickle-cell disease, Tay-Sachs disease, Niemann-Pick disease,spinal muscular atrophy, or Roberts syndrome can comprise a copy of agene that comprises an exon that when properly transcribed into fullyprocessed mRNA can encode the full-length functional for of the protein,can comprises a copy of a gene that can comprise a copy of a gene thatcomprises a set of exons that when properly transcribed into fullyprocessed mRNA can encode the full-length functional form of theprotein, or can comprise a defective copy of the gene, which can beincapable of producing a full-length functional form of the protein.

Exemplary X-linked dominant disorder can include X-linkedhypophosphatemic rickets, Rett syndrome, incontinentia pigmenti type 2,Aicardi syndrome, or Klinefelter syndrome.

An X-linked dominant disorder such as: X-linked hypophosphatemicrickets, Rett syndrome, incontinentia pigmenti type 2, Aicardi syndrome,or Klinefelter syndrome can be characterized by an impaired productionof a protein or by a defective splicing. An X-linked dominant disordersuch as: X-linked hypophosphatemic rickets, Rett syndrome, incontinentiapigmenti type 2, Aicardi syndrome, or Klinefelter syndrome can comprisea copy of a gene that comprises an exon that when properly transcribedinto fully processed mRNA can encode the full-length functional for ofthe protein, can comprises a copy of a gene that can comprise a copy ofa gene that comprises a set of exons that when properly transcribed intofully processed mRNA can encode the full-length functional form of theprotein, or can comprise a defective copy of the gene, which can beincapable of producing a full-length functional form of the protein.

Exemplary X-linked recessive disorder can include hemophilia A, Duchennemuscular dystrophy, Lesch-Nyhan syndrome, or Turner syndrome.

An X-linked recessive disorder such as: hemophilia A, Duchenne musculardystrophy, Lesch-Nyhan syndrome, or Turner syndrome can be characterizedby an impaired production of a protein or by a defective splicing. AnX-linked recessive disorder such as: hemophilia A, Duchenne musculardystrophy, Lesch-Nyhan syndrome, or Turner syndrome can comprise a copyof a gene that comprises an exon that when properly transcribed intofully processed mRNA can encode the full-length functional for of theprotein, can comprises a copy of a gene that can comprise a copy of agene that comprises a set of exons that when properly transcribed intofully processed mRNA can encode the full-length functional form of theprotein, or can comprise a defective copy of the gene, which can beincapable of producing a full-length functional form of the protein.

Exemplary Y-linked disorder can include Swyer syndrome or a form ofretinitis pigmentosa.

A Y-linked disorder such as Swyer syndrome or a form of retinitispigmentosa can be characterized by an impaired production of a proteinor by a defective splicing. A Y-linked disorder such as Swyer syndromeor a form of retinitis pigmentosa can comprise a copy of a gene thatcomprises an exon that when properly transcribed into fully processedmRNA can encode the full-length functional for of the protein, cancomprises a copy of a gene that can comprise a copy of a gene thatcomprises a set of exons that when properly transcribed into fullyprocessed mRNA can encode the full-length functional form of theprotein, or can comprise a defective copy of the gene, which can beincapable of producing a full-length functional form of the protein.

Exemplary mitochondrial disease can include Leber's hereditary opticneuropathy.

Mitochondrial disease such as Leber's hereditary optic neuropathy can becharacterized by an impaired production of a protein or by a defectivesplicing. Mitochondrial disease such as Leber's hereditary opticneuropathy can comprise a copy of a gene that comprises an exon thatwhen properly transcribed into fully processed mRNA can encode thefull-length functional for of the protein, can comprises a copy of agene that can comprise a copy of a gene that comprises a set of exonsthat when properly transcribed into fully processed mRNA can encode thefull-length functional form of the protein, or can comprise a defectivecopy of the gene, which can be incapable of producing a full-lengthfunctional form of the protein.

Exemplary multifactorial or polygenic disorder can include heartdisease, diabetes, autoimmune diseases such as multiple sclerosis,inflammatory bowel disease, or cancer.

Multifactorial or polygenic disorder such as heart disease, diabetes,autoimmune diseases such as multiple sclerosis, inflammatory boweldisease, or cancer can be characterized by an impaired production of aprotein or by a defective splicing. Multifactorial or polygenic disordersuch as heart disease, diabetes, autoimmune diseases such as multiplesclerosis, inflammatory bowel disease, or cancer can comprise a copy ofa gene that comprises an exon that when properly transcribed into fullyprocessed mRNA can encode the full-length functional for of the protein,can comprises a copy of a gene that can comprise a copy of a gene thatcomprises a set of exons that when properly transcribed into fullyprocessed mRNA can encode the full-length functional form of theprotein, or can comprise a defective copy of the gene, which can beincapable of producing a full-length functional form of the protein.

In some instances, compositions and methods described herein is used totreat a genetic disorder or condition such as a hereditary disease.Compositions and methods described herein can be used to treat a geneticdisorder or condition such as a hereditary disease that is characterizedby an impaired production of a protein. Compositions and methodsdescribed herein can be used to treat a genetic disorder or conditionsuch as a hereditary disease that is characterized by a defectivesplicing.

Compositions and methods described herein can also be used to treat agenetic disorder or condition such as an autosomal dominant disorder, anautosomal recessive disorder, X-linked dominant disorder, X-linkedrecessive disorder, Y-linked disorder, mitochondrial disease, ormultifactorial or polygenic disorder. Compositions and methods describedherein can be used to treat an autosomal dominant disorder, an autosomalrecessive disorder, X-linked dominant disorder, X-linked recessivedisorder, Y-linked disorder, mitochondrial disease, or multifactorial orpolygenic disorder, in which the disorder or condition is characterizedby an impaired production of a protein. Compositions and methodsdescribed herein can also be used to treat an autosomal dominantdisorder, an autosomal recessive disorder, X-linked dominant disorder,X-linked recessive disorder, Y-linked disorder, mitochondrial disease,or multifactorial or polygenic disorder, in which the disorder orcondition is characterized by a defective splicing.

In some instances, a disease or condition includes muscular dystrophy,spinal muscular atrophy (SMA), ataxia-telangiectasia, X-linkedagammaglobulinaemia, diabetes, or cancer.

Muscular dystrophy is a group of muscle diseases that can weaken themusculoskeletal system and can hamper locomotion. It can becharacterized by the progressive skeletal muscle weakness, defects inmuscle proteins, and the death of muscle cells and tissues. One commonform of muscular dystrophy can be Duchenne muscular dystrophy (DMD).Additional forms of muscular dystrophy can include Becker, limb-girdle,congenital, facioscapulohumeral, myotonic, oculopharyngeal, distal, andEmery-Dreifuss muscular dystrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder that isone of the most common genetic causes of childhood mortality. The maincharacteristic of the disease can be a progressive loss of spinal cordmotor neurons, resulting in skeletal muscle denervation with subsequentweakness, atrophy, and paralysis of voluntary muscles. The SMA locus canbe mapped to a complex inverted repeat of ˜500 kb on Chromosome 5q13that contains several genes. The main cause of SMA can be homozygousloss of the telomeric copy of the survivor of motor neuron gene (SMN1)located within the inverted repeat. A duplicated gene within thecentromeric copy of the inverted repeat (SMN2) can also be transcribed,but the SMN2 gene does not completely compensate for loss of SMN1function.

Ataxia-telangiectasia (A-T) or Louis-Bar syndrome is a rareneurodegenerative inherited disease. The term “ataxia” refers to poorcoordination and the term “telangiectasia” refers to small dilated bloodvessels. Both terms characterize the hallmarks of this disease. AT canimpair the cerebellum and additional areas of the brain which can causeimpaired movement and coordination. AT can also weaken the immune systemthereby increasing infection and can impair DNA repair, thereby increasethe risk of cancer. A-T can be associated with a defect in the gene ATM,which is responsible for managing cellular response to multiple form ofstress.

X-linked agammaglobulinaemia, also known as X-linkedhypogammaglobulinemia. XLA, Bruton type agammaglobulinemia, Brutonsyndrome, or Sex-linked agammaglobulinemia, is an X-linked geneticdisorder that can affect the body's ability to fight infection. XLApatients lack mature B cells and as a result, lack the necessaryantibodies to combat infection. Bruton's tyrosine kinase (BTK) can beassociated with mediating B cell development and maturation and the BTKgene can be associated with XLA.

Diabetes mellitus (DM) (commonly known as diabetes) is a group ofmetabolic diseases characterized by a high blood sugar level over aprolonged period. Symptoms of diabetes can include weight loss, polyuriaor increased urination, polydipsia or increased thirst, and polyphagiaor increased hunger. Diabetes can be classified into four categories:type 1, type 2, gestational diabetes, and other specific types ofdiabetes. Type 1 diabetes can be characterized by a loss ofinsulin-producing beta cells of the islets of Langerhans in thepancreas, leading to insulin deficiency. Type 2 diabetes can becharacterized by insulin resistance, which can also be combined with areduced insulin secretion. Gestational diabetes can resemble type 2diabetes, and can involve combination of inadequate insulin secretionand responsiveness. Other specific types of diabetes can includeprediabetes, latent autoimmune diabetes of adults (LADA) and congenitaldiabetes.

Cancer can be a solid tumor or a hematologic malignancy. A solid tumorcan be a sarcoma or a carcinoma. Sarcoma can be a cancer of bone,cartilage, fat muscle, vascular or hematopoietic tissues. Exemplarysarcoma can include alveolar rhabdomyosarcoma, alveolar soft partsarcoma, ameloblastoma, angiosarcoma, chondrosarcoma, chordoma, clearcell sarcoma of soft tissue, dedifferentiated liposarcoma, desmoid,desmoplastic small round cell tumor, embryonal rhabdomyosarcoma,epithelioid fibrosarcoma, epithelioid hemangioendothelioma, epithelioidsarcoma, esthesioneuroblastoma, Ewing sarcoma, extrarenal rhabdoidtumor, extraskeletal myxoid chondrosarcoma, extraskeletal osteosarcoma,fibrosarcoma, giant cell tumor, hemangiopericytoma, infantilefibrosarcoma, inflammatory myofibroblastic tumor, Kaposi sarcoma,leiomyosarcoma of bone, liposarcoma, liposarcoma of bone, malignantfibrous histiocytoma (MFH), malignant fibrous histiocytoma (MFH) ofbone, malignant mesenchymoma, malignant peripheral nerve sheath tumor,mesenchymal chondrosarcoma, myxofibrosarcoma, myxoid liposarcoma,myxoinflammatory fibroblastic sarcoma, neoplasms with perivascularepitheioid cell differentiation, osteosarcoma, parosteal osteosarcoma,neoplasm with perivascular epitheioid cell differentiation, periostealosteosarcoma, pleomorphic liposarcoma, pleomorphic rhabdomyo sarcoma,PNET/extraskeletal Ewing tumor, rhabdomyosarcoma, round cellliposarcoma, small cell osteosarcoma, solitary fibrous tumor, synovialsarcoma, telangiectatic osteosarcoma.

Carcinoma can be a cancer developed from epithelial cells. Exemplarycarcinoma can include adenocarcinoma, squamous cell carcinoma,adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma,small cell carcinoma, anal cancer, appendix cancer, bile duct cancer(i.e., cholangiocarcinoma), bladder cancer, brain tumor, breast cancer,cervical cancer, colon cancer, cancer of Unknown Primary (CUP),esophageal cancer, eye cancer, fallopian tube cancer,gastroenterological cancer, kidney cancer, liver cancer, lung cancer,medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreaticcancer, parathyroid disease, penile cancer, pituitary tumor, prostatecancer, rectal cancer, skin cancer, stomach cancer, testicular cancer,throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvarcancer.

Hematologic malignancy is a malignancy of the blood system and caninclude T-cell based and B-cell based malignancies. Exemplaryhematologic malignancy can include myeloid leukaemia, myeloproliferativeneoplasias, peripheral T-cell lymphoma not otherwise specified(PTCL-NOS), anaplastic large cell lymphoma, angioimmunoblastic lymphoma,cutaneous T-cell lymphoma, adult T-cell leukemia/lymphoma (ATLL),blastic NK-cell lymphoma, enteropathy-type T-cell lymphoma,hematosplenic gamma-delta T-cell lymphoma, lymphoblastic lymphoma, nasalNK/T-cell lymphomas, treatment-related T-cell lymphomas, chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high riskCLL, non-CLL/SLL lymphoma, prolymphocytic leukemia (PLL), follicularlymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle celllymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma,extranodal marginal zone B cell lymphoma, nodal marginal zone B celllymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma,primary mediastinal B-cell lymphoma (PMBL), immunoblastic large celllymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocyticleukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma,plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B celllymphoma, intravascular large B cell lymphoma, primary effusionlymphoma, or lymphomatoid granulomatosis.

In some instances, compositions and methods described herein is used totreat muscular dystrophy, spinal muscular atrophy (SMA),ataxia-telangiectasia, X-linked agammaglobulinaemia, diabetes, orcancer. Compositions and methods described herein can be used to treatmuscular dystrophy, spinal muscular atrophy (SMA),ataxia-telangiectasia, X-linked agammaglobulinaemia, diabetes, or cancerin which the disease or disorder is associated with an impairedproduction of a protein. Compositions and methods described herein canbe used to treat muscular dystrophy, spinal muscular atrophy (SMA),ataxia-telangiectasia, X-linked agammaglobulinaemia, diabetes, or cancerin which the disease or disorder is characterized by a defectivesplicing.

The disease may be any genetic condition caused by mutations leading toretention of entire introns in mature transcripts. The disease may bediabetes. The disease may be diabetes type I. The disease may bediabetes type II. In another embodiment, the disease may be cancer. Thecancer may be myeloid leukaemia or myeloproliferative neoplasias. Thecancer may sustain mutations in any of the spliceosomal components thatfacilitate recognition of 3′ splice sites. The polynucleic acid polymermay be used to increase endogenous expression in a subject with residualβ-cell activity. The polynucleic acid polymer may be used to increaseexpression of proinsulin in other diabetes patients, including those whoreceived transplanted β-cells. The polynucleic acid polymer may be usedas antisense therapy of malignant tumours containing mutations in genesencoding U2 components (e.g., >20% of myeloid leukaemias).

In an embodiment where the disease is diabetes, reducing the incidenceof intron retention may be during fetal development of the subject. Forexample a pregnant mother may be administered with the polynucleic acidpolymer in order to reduce the intron retention in the fetus.

The subject may be eukaryote. The subject may be mammalian. The subjectmay be human. The subject may be a non-human primate. The subject may bea non-primate mammal such as a rat, mouse, ferret, dog, cat, or pig. Thesubject may be a fetus, such as a human fetus.

The method may comprise a step of determining if a disease pathology iscaused by an intron retention in a gene transcript prior to treatment.The determination may use any suitable assay or genetic analysisavailable to the skilled person.

In some instances, detection is done at a nucleic acid level withnucleic acid-based techniques such as in situ hybridization and RT-PCR.Sequencing technologies can include next-generation sequencingtechnologies such as Helicos True Single Molecule Sequencing (tSMS)(Harris T. D. et al. (2008) Science 320:106-109); 454 sequencing (Roche)(Margulies, M. et al. 2005, Nature, 437, 376-380); SOLiD technology(Applied Biosystems); SOLEXA sequencing (Illumina); single molecule,real-time (SMRT™) technology of Pacific Biosciences; nanopore sequencing(Soni G V and Meller A. (2007) Clin Chem 53: 1996-2001); semiconductorsequencing (Ion Torrent; Personal Genome Machine); DNA nanoballsequencing; sequencing using technology from Dover Systems (Polonator),and technologies that do not require amplification or otherwisetransform native DNA prior to sequencing (e.g., Pacific Biosciences andHelicos), such as nanopore-based strategies (e.g. Oxford Nanopore, GeniaTechnologies, and Nabsys). Sequencing technologies can also includeSanger sequencing, Maxam-Gilbert sequencing, Shotgun sequencing, bridgePCR, mass spectrometry based sequencing, microfluidic based Sangersequencing, microscopy-based sequencing, RNAP sequencing, orhybridization based sequencing.

Sequencing of a gene transcript of interest may also include anamplification step. Exemplary amplification methodologies include, butare not limited to, polymerase chain reaction (PCR), nucleic acidsequence based amplification (NASBA), self-sustained sequencereplication (3SR), loop mediated isothermal amplification (LAMP), stranddisplacement amplification (SDA), whole genome amplification, multipledisplacement amplification, strand displacement amplification, helicasedependent amplification, nicking enzyme amplification reaction,recombinant polymerase amplification, reverse transcription PCR,ligation mediated PCR, or methylation specific PCR.

Additional methods that can be used to obtain a nucleic acid sequenceinclude, e.g., whole-genome RNA expression array, enzyme-linkedimmunosorbent assay (ELISA), genome sequencing, de novo sequencing,Pacific Biosciences SMRT sequencing, immunohistochemistry (IHC),immunoctyochemistry (ICC), mass spectrometry, tandem mass spectrometry,matrix-assisted laser desorption ionization time of flight massspectrometry (MALDI-TOF MS), in-situ hybridization, fluorescent in-situhybridization (FISH), chromogenic in-situ hybridization (CISH), silverin situ hybridization (SISH), digital PCR (dPCR), reverse transcriptionPCR, quantitative PCR (Q-PCR), single marker qPCR, real-time PCR,nCounter Analysis (Nanostring technology), Western blotting, Southernblotting, SDS-PAGE, gel electrophoresis, and Northern blotting.

In some cases, detection can be done at a protein level, using, forexample, immunoprecipitation based assays such as Western blot, orELISA. Additionally, methods such as electrophoresis and massspectrometry analysis can also be utilized for detection of a protein ofinterest.

According to another aspect of the invention, there is provided a methodof modulating intron splicing in a cell, comprising hybridizing apolynucleic acid polymer to a region of pre-mRNA, wherein the regioncomprises or consists of SEQ ID NO: 46, or optionally, a region havingat least 95% identity to SEQ ID NO: 46. The region may have at least 98%or 99% identity to SEQ ID NO: 46.

According to another aspect of the invention, there is provided a methodof modulating intron splicing in a cell, comprising hybridizing apolynucleic acid polymer to a region of pre-mRNA, wherein the regioncomprises or consists of SEQ ID NO: 3, or optionally, a region having atleast 95% identity to SEQ ID NO: 3. The region may have at least 98% or99% identity to SEQ ID NO: 3.

According to another aspect of the invention, there is provided a methodof modulating intron splicing in a cell, comprising hybridizing apolynucleic acid polymer to a region of pre-mRNA, wherein the regioncomprises or consists of a sequence complementary to any of the group ofsequences comprising SEQ ID NOs: 47 to 434; or combinations thereof.

According to another aspect of the invention, there is provided a methodof modulating intron splicing in a cell, comprising hybridizing apolynucleic acid polymer to a region of pre-mRNA, wherein the regioncomprises or consists of a sequence complementary to a sequence havingat least 95% identity to any of the group of sequences comprising SEQ IDNOs: 47 to 434; or combinations thereof. The region may comprise orconsist of a sequence complementary to a sequence having at least 98%identity to any of the group of sequences comprising SEQ ID NOs: 47 to434; or combinations thereof. The region may comprise or consist of asequence complementary to a sequence having at least 99% identity to anyof the group of sequences comprising SEQ ID NOs: 47 to 434; orcombinations thereof.

The cell may be in vitro. The cell may be ex vivo. The cell may be aeukaryotic cell or a prokaryotic cell. The cell may be a eukaryoticcell. The cell may be a mammalian cell from human; non-human primate; ornon-primate mammals such as cat, rat, mouse, dog, ferret, or pigs. Thecell may be a human cell such as from an epithelial cell, connectivetissue cell, hormone secreting cell, a nerve cell, a skeletal musclecell, a blood cell, or an immune system cell. The cell may be a tumorcell such as a solid tumor cell or a hematologic malignant cell.

According to another aspect of the invention, there is providedpolynucleic acid polymer which is antisense to at least part of a regionof polynucleic acid polymer comprising or consisting of SEQ ID NO: 46,or optionally a region of polynucleic acid polymer comprising orconsisting of a sequence having at least 95% sequence identity to SEQ IDNO: 46. The region may have at least 98% or 99% identity to SEQ ID NO:46.

According to another aspect of the invention, there is providedpolynucleic acid polymer which is antisense to at least part of a regionof polynucleic acid polymer comprising or consisting of SEQ ID NO: 3, oroptionally a region of polynucleic acid polymer comprising or consistingof a sequence having at least 95% sequence identity to SEQ ID NO: 3. Theregion may have at least 98% or 99% identity to SEQ ID NO: 3.

According to another aspect of the invention, there is providedpolynucleic acid polymer which is antisense to at least part of a regionof polynucleic acid polymer, wherein the region comprises or consists ofa sequence complementary to any of the group of sequences comprising SEQID NOs: 47 to 434; or combinations thereof.

According to another aspect of the invention, there is providedpolynucleic acid polymer which is antisense to at least part of a regionof polynucleic acid polymer, wherein the region comprises or consists ofa sequence complementary to any of the group of sequences comprising SEQID NOs: 47 to 434; or combinations thereof; or optionally a region ofpolynucleic acid polymer comprising or consisting of a sequence havingat least 95% sequence identity to SEQ ID NOs: 47 to 434. The region mayhave at least 98% or 99% identity to SEQ ID SEQ ID NOs: 47 to 434.

Reference to being antisense to at least part of a region of polynucleicacid polymer may be understood by the skilled person to mean a region ofat least 5 consecutive nucleotides. Reference to being antisense to atleast part of a region of polynucleic acid polymer may be understood bythe skilled person to mean a region of at least 10 consecutivenucleotides.

According to another aspect of the invention, there is providedpolynucleic acid polymer comprising or consisting of a nucleic acidsequence selected from any of the group comprising SEQ ID NO: 1; SEQ IDNO: 2; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 8; SEQ IDNO: 10; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 16; SEQID NO: 17; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23;SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO:31; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 37; SEQ IDNO: 38; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 43; and SEQ ID NO: 44;or combinations thereof.

According to another aspect of the invention, there is providedpolynucleic acid polymer comprising or consisting of a nucleic acidsequence having at least 99% identity to a sequence selected from any ofthe group comprising SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ IDNO: 5; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 11; SEQ IDNO: 13; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 19; SEQID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 26;SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO:34; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 40; SEQ IDNO: 41; SEQ ID NO: 43; and SEQ ID NO: 44; or combinations thereof.According to another aspect of the invention, there is providedpolynucleic acid polymer comprising or consisting of a nucleic acidsequence having at least 98% identity to a sequence selected from any ofthe group comprising SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ IDNO: 5; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 11; SEQ IDNO: 13; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 19; SEQID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 26;SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO:34; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 40; SEQ IDNO: 41; SEQ ID NO: 43; and SEQ ID NO: 44; or combinations thereof.According to another aspect of the invention, there is providedpolynucleic acid polymer comprising or consisting of a nucleic acidsequence having at least 95% identity to a sequence selected from any ofthe group comprising SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 4; SEQ IDNO: 5; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 11; SEQ IDNO: 13; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 19; SEQID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 26;SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO:34; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 40; SEQ IDNO: 41; SEQ ID NO: 43; and SEQ ID NO: 44; or combinations thereof.

According to another aspect of the invention, there is providedpolynucleic acid polymer comprising or consisting of a nucleic acidsequence selected from any of the group comprising SEQ ID NOs: 47 to434; or combinations thereof. It is understood that uracil nucleotidesmay be substituted with thymine nucleotides (e.g. the DNA form of theRNA of such sequences).

According to another aspect of the invention, there is providedpolynucleic acid polymer comprising or consisting of a nucleic acidsequence having at least 99% identity to a sequence selected from any ofthe group comprising SEQ ID NOs: 47 to 434; or combinations thereof.According to another aspect of the invention, there is providedpolynucleic acid polymer comprising or consisting of a nucleic acidsequence having at least 98% identity to a sequence selected from any ofthe group comprising SEQ ID NOs: 47 to 434; or combinations thereof.According to another aspect of the invention, there is providedpolynucleic acid polymer comprising or consisting of a nucleic acidsequence having at least 95% identity to a sequence selected from any ofthe group comprising SEQ ID NOs: 47 to 434; or combinations thereof. Itis understood that uracil nucleotides may be substituted with thyminenucleotides (e.g. the DNA form of the RNA of such sequences).

The polynucleic acid polymer may be isolated polynucleic acid polymer.The polynucleic acid polymer may be conjugated to, or bound by, adelivery vehicle suitable for delivering the polynucleic acid polymer tocells. The delivery vehicle may be capable of site specific, tissuespecific or cell specific delivery. For example, the delivery vehiclemay be a cell specific viral particle, or component thereof,alternatively, the delivery vehicle may be a cell specific antibodyparticle, or component thereof. The polynucleic acid polymer may betargeted for delivery to beta cells in the pancreas. The polynucleicacid polymer may be targeted for delivery to thymic cells. Thepolynucleic acid polymer may be targeted for delivery to malignantcells. The polynucleic acid polymer may be modified to increase itsstability. In an embodiment where the polynucleic acid polymer is RNAthe polynucleic acid polymer may be modified to increase its stability.The polynucleic acid polymer may be modified by 2′-O-methyl and2′-O-methoxyethyl ribose. Suitable modifications to the RNA to increasestability for delivery will be apparent to the skilled person.

The polynucleic acid polymer may be part of a plasmid vector. Thepolynucleic acid polymer may be part of a viral vector. The polynucleicacid polymer may be encoded on a viral vector.

According to another aspect of the invention, there is provided a vectorcomprising the polynucleic acid polymer of the invention. The vector maycomprise a viral vector. The viral vector may comprise adeno-associatedviral vector. The vector may comprise any virus that targets thepolynucleic acid polymer to malignant cells or specific cell type.

According to another aspect of the invention, there is provided adelivery vehicle comprising, or bound to, the polynucleic acid polymerof the invention. The delivery vehicle may comprise a lipid-basednanoparticle; a cationic cell penetrating peptide (CPP); or a linear orbranched cationic polymer; or a bioconjugate, such as cholesterol, bileacid, lipid, peptide, polymer, protein, or an aptamer, which isconjugated to the polynucleic acid polymer for intracellular deliveryand/or improved stability. The delivery vehicle may be cell or tissuespecific or developmental stage specific. The delivery vehicle maycomprise an antibody, or part thereof. The antibody may be specific fora cell surface marker on the cell of interest for delivery of thepolynucleic acid polymer to the specific cell. For example, the antibodymay comprise an anti-GAD antibody for targeted non-viral polynucleicacid polymer delivery to islet beta cells according to Ji Hoon Jeong etal (Journal of Controlled Release 107 (2005) 562-570), incorporatedherein by reference. For example, the anti-GAD antibody conjugatinganti-GAD Fab′ fragment to PEI via a PEG linker (PEI-PEG-Fab′). Otherspecific antibodies may be used in such a conjugation for targetedtissue delivery of the polynucleic acid polymer.

Pharmaceutical Composition/Formulations, Dosing, and Treatment Regimens

According to one aspect of the invention, there is provided atherapeutic agent for the treatment of a disease or conditioncharacterized by impaired production of a functional form of a proteinwhich comprises administering to the subject a pharmaceuticalcomposition comprising a therapeutic agent that induces an increase insplicing out of an intron in a partially processed mRNA transcript,wherein the subject has a pool of partially processed mRNA transcripts,which are capable of encoding copies of the full-length functional formof the protein and each of which comprise at least one retained intronthat inhibits translation of the partially processed mRNA transcripts;and contacting a target cell of the subject with the therapeutic agentto induce a portion of the pool of the partially processed mRNAtranscripts to undergo splicing to remove the at least one retainedintron from each of the partially processed mRNA transcripts in theportion, to produce fully processed mRNA transcripts, wherein the fullyprocessed mRNA transcripts are translated to express copies of thefull-length functional form of the protein, which treat the disease orcondition.

The therapeutic agent can causes activation of one or more splicingprotein complexes in the cell to remove the at least one retained intronfrom each of the partially processed mRNA transcripts in the portion ofthe pool of the partially processed mRNA transcripts. The therapeuticagent can inhibit a protein that regulates intron splicing activity. Thetherapeutic agent can activate a protein that regulates intron splicingactivity. The therapeutic agent may interact or bind to a protein thatregulates intron splicing activity. The therapeutic agent may interactor bind to target polynucleotide sequence of the partially processedmRNA transcripts. In some embodiments the therapeutic agent can be apolynucleic acid polymer, such as the polynucleic acid polymersdescribed herein. In some embodiments, the therapeutic agent can be asmall molecule.

The small molecule can be a molecule of less than 900 Daltons, and caninitiate one or more splicing protein complexes in the cell to removethe at least one retained intron from each of the partially processedmRNA transcripts in the portion of the pool of the partially processedmRNA transcripts. The small molecule can inhibit a protein thatregulates intron splicing activity. The small molecule can activate aprotein that regulates intron splicing activity. The small molecule mayinteract or bind to a protein that regulates intron splicing activity,or may interact or bind to target polynucleotide sequence of thepartially processed mRNA transcripts.

According to another aspect of the invention, there is provided acomposition comprising the polynucleic acid polymer of the invention.The composition may be a pharmaceutically acceptable composition. Thecomposition may comprise a pharmaceutically acceptable carrier. Thecomposition may comprise an additional active agent, such as a drug orpro-drug. The composition may comprise combinations of differentpolynucleic acid polymers, such as SSOs, for therapy.

The composition may comprise at least one other biologically activemolecule in addition to the polynucleic acid polymer. The biologicallyactive molecule may be drug or a pro-drug. The biologically activemolecule may comprise nucleic acid or amino acid. The biologicallyactive molecule may comprise a small molecule (e.g. a molecule of <900Daltons).

A pharmaceutical composition described herein may comprise a polynucleicacid polymer that hybridizes to a target sequence of a partiallyprocessed mRNA transcript which encodes a protein and which comprises aretained intron, wherein the target sequence is in between two Gquadruplexes, wherein the polynucleic acid polymer is capable ofinducing splicing out of the retained intron from the partiallyprocessed mRNA transcript; and a pharmaceutically acceptable excipientand/or a delivery vehicle. The pharmaceutical composition describedherein may also comprise a polynucleic acid polymer that hybridizes to atarget sequence of a partially processed mRNA transcript which encodes aprotein and which comprises a retained intron, wherein the polynucleicacid polymer hybridizes to an intronic splicing regulatory element ofthe partially processed mRNA transcript, wherein the intronic splicingregulatory element comprises a first CCC motif, and wherein thepolynucleic acid polymer is capable of inducing splicing out of theretained intron from the partially processed mRNA transcript; and apharmaceutically acceptable excipient and/or a delivery vehicle. Inaddition, the pharmaceutical composition described herein may comprise apolynucleic acid polymer that hybridizes to a target sequence of apartially processed mRNA transcript which encodes a protein and whichcomprises a retained intron, wherein the polynucleic acid polymerhybridizes to a binding motif of the partially processed mRNAtranscript, wherein the binding motif does not form a G quadruplex, andwherein the polynucleic acid polymer is capable of inducing splicing outof the retained intron from the partially processed mRNA transcript; anda pharmaceutically acceptable excipient and/or a delivery vehicle.

A pharmaceutical composition described herein may further comprise apolynucleic acid polymer that hybridizes to a target sequence of apartially processed mRNA transcript which encodes a protein and whichcomprises a retained intron, wherein the polynucleic acid polymerhybridizes to a binding motif of the partially processed mRNAtranscript, and wherein the binding motif forms a hairpin structure,wherein the polynucleic acid polymer is capable of inducing splicing outof the retained intron from the partially processed mRNA transcript; anda pharmaceutically acceptable excipient and/or a delivery vehicle.

A pharmaceutical formulations described herein can be administered to asubject by multiple administration routes, including but not limited to,parenteral (e.g., intravenous, subcutaneous, intramuscular), oral,intranasal, buccal, topical, rectal, or transdermal administrationroutes. In some instances, the pharmaceutical composition describeherein is formulated for parenteral (e.g., intravenous, subcutaneous,intramuscular) administration. In other instances, the pharmaceuticalcomposition describe herein is formulated for oral administration. Instill other instances, the pharmaceutical composition describe herein isformulated for intranasal administration.

A pharmaceutical formulations described herein may include, but are notlimited to, aqueous liquid dispersions, self-emulsifying dispersions,solid solutions, liposomal dispersions, aerosols, solid dosage forms,powders, immediate release formulations, controlled releaseformulations, fast melt formulations, tablets, capsules, pills, delayedrelease formulations, extended release formulations, pulsatile releaseformulations, multiparticulate formulations, and mixed immediate andcontrolled release formulations.

A pharmaceutical formulations may include a carrier or carrier materialswhich may include any commonly used excipients in pharmaceutics andshould be selected on the basis of compatibility with the compositiondisclosed herein, and the release profile properties of the desireddosage form. Exemplary carrier materials include, e.g., binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, diluents, and thelike. Pharmaceutically compatible carrier materials may include, but arenot limited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerine, magnesiumsilicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters,sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine,sodium chloride, tricalcium phosphate, dipotassium phosphate, celluloseand cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like. See,e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed(Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999).

A pharmaceutical formulations may include dispersing agents, and/orviscosity modulating agents which may include materials that control thediffusion and homogeneity of a drug through liquid media or agranulation method or blend method. In some embodiments, these agentsalso facilitate the effectiveness of a coating or eroding matrix.Exemplary diffusion facilitators/dispersing agents include, e.g.,hydrophilic polymers, electrolytes, Tween® 60 or 80, PEG,polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and thecarbohydrate-based dispersing agents such as, for example, hydroxypropylcelluloses (e.g., HPC, HPC-SL, and HPC-L), hydroxypropylmethylcelluloses (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M),carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate,hydroxypropylmethylcellulose acetate stearate (HPMCAS), noncrystallinecellulose, magnesium aluminum silicate, triethanolamine, polyvinylalcohol (PVA), vinyl pyrrolidone/vinyl acetate copolymer (S630),4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol), poloxamers (e.g., PluronicsF68®, F88®, and F108®, which are block copolymers of ethylene oxide andpropylene oxide); and poloxamines (e.g., Tetronic 908®, also known asPoloxamine 908®, which is a tetrafunctional block copolymer derived fromsequential addition of propylene oxide and ethylene oxide toethylenediamine (BASF Corporation, Parsippany, N.J.)),polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetatecopolymer (S-630), polyethylene glycol, e.g., the polyethylene glycolcan have a molecular weight of about 300 to about 6000, or about 3350 toabout 4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, polysorbate-80, sodium alginate, gums, such as, e.g.,gum tragacanth and gum acacia, guar gum, xanthans, including xanthangum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose,methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodiumalginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitanmonolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates,chitosans and combinations thereof. Plasticizers such as cellulose ortriethyl cellulose can also be used as dispersing agents. Dispersingagents particularly useful in liposomal dispersions and self-emulsifyingdispersions are dimyristoyl phosphatidyl choline, natural phosphatidylcholine from eggs, natural phosphatidyl glycerol from eggs, cholesteroland isopropyl myristate.

A pharmaceutical formulations may include pH adjusting agents orbuffering agents which may include acids such as acetic, boric, citric,lactic, phosphoric and hydrochloric acids; bases such as sodiumhydroxide, sodium phosphate, sodium borate, sodium citrate, sodiumacetate, sodium lactate and tris-hydroxymethylaminomethane; and bufferssuch as citrate/dextrose, sodium bicarbonate and ammonium chloride. Suchacids, bases and buffers are included in an amount required to maintainpH of the composition in an acceptable range.

A pharmaceutical formulation may also include one or more salts in anamount required to bring osmolality of the composition into anacceptable range. Such salts may include those having sodium, potassiumor ammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

A pharmaceutical formulations may further include diluent which may alsobe used to stabilize compounds because they can provide a more stableenvironment. Salts dissolved in buffered solutions (which also canprovide pH control or maintenance) can be utilized as diluents in theart, including, but not limited to a phosphate buffered saline solution.In certain instances, diluents increase bulk of the composition tofacilitate compression or create sufficient bulk for homogenous blendfor capsule filling. Such compounds can include e.g., lactose, starch,mannitol, sorbitol, dextrose, microcrystalline cellulose such asAvicel®; dibasic calcium phosphate, dicalcium phosphate dihydrate;tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-driedlactose; pregelatinized starch, compressible sugar, such as Di-Pac®(Amstar); mannitol, hydroxypropylmethylcellulose,hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents,confectioner's sugar; monobasic calcium sulfate monohydrate, calciumsulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzedcereal solids, amylose; powdered cellulose, calcium carbonate; glycine,kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

A pharmaceutical formulations may include disintegration agents ordisintegrants to facilitate the breakup or disintegration of asubstance. The term “disintegrate” can include both the dissolution anddispersion of the dosage form when contacted with gastrointestinalfluid. Examples of disintegration agents can include a starch, e.g., anatural starch such as corn starch or potato starch, a pregelatinizedstarch such as National 1551 or Amijel®, or sodium starch glycolate suchas Promogel® or Explotab®, a cellulose such as a wood product,methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel®PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, andSolka-Floc®, methylcellulose, croscarmellose, or a cross-linkedcellulose, such as cross-linked sodium carboxymethylcellulose(Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linkedcroscarmellose, a cross-linked starch such as sodium starch glycolate, across-linked polymer such as crospovidone, a cross-linkedpolyvinylpyrrolidone, alginate such as alginic acid or a salt of alginicacid such as sodium alginate, a clay such as Veegum® HV (magnesiumaluminum silicate), a gum such as agar, guar, locust bean, Karaya,pectin, or tragacanth, sodium starch glycolate, bentonite, a naturalsponge, a surfactant, a resin such as a cation-exchange resin, citruspulp, sodium lauryl sulfate, sodium lauryl sulfate in combinationstarch, and the like.

A pharmaceutical formulations may include filling agents such aslactose, calcium carbonate, calcium phosphate, dibasic calciumphosphate, calcium sulfate, microcrystalline cellulose, cellulosepowder, dextrose, dextrates, dextran, starches, pregelatinized starch,sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride,polyethylene glycol, and the like.

A pharmaceutical formulations may include flavoring agents and/orsweeteners” such as for example, acacia syrup, acesulfame K, alitame,anise, apple, aspartame, banana, Bavarian cream, berry, black currant,butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream,chocolate, cinnamon, bubble gum, citrus, citrus punch, citrus cream,cotton candy, cocoa, cola, cool cherry, cool citrus, cyclamate,cylamate, dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger,glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit, honey,isomalt, lemon, lime, lemon cream, monoammonium glyrrhizinate(MagnaSweet®), maltol, mannitol, maple, marshmallow, menthol, mintcream, mixed berry, neohesperidine DC, neotame, orange, pear, peach,peppermint, peppermint cream, Prosweet® Powder, raspberry, root beer,rum, saccharin, safrole, sorbitol, spearmint, spearmint cream,strawberry, strawberry cream, stevia, sucralose, sucrose, sodiumsaccharin, saccharin, aspartame, acesulfame potassium, mannitol, talin,sylitol, sucralose, sorbitol, Swiss cream, tagatose, tangerine,thaumatin, tutti fruitti, vanilla, walnut, watermelon, wild cherry,wintergreen, xylitol, or any combination of these flavoring ingredients,e.g., anise-menthol, cherry-anise, cinnamon-orange, cherry-cinnamon,chocolate-mint, honey-lemon, lemon-lime, lemon-mint, menthol-eucalyptus,orange-cream, vanilla-mint, and mixtures thereof.

Lubricants and glidants may also be included in the pharmaceuticalformulations described herein which can prevent, reduce or inhibitadhesion or friction of materials. Exemplary lubricants can include,e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, ahydrocarbon such as mineral oil, or hydrogenated vegetable oil such ashydrogenated soybean oil (Sterotex®), higher fatty acids and theiralkali-metal and alkaline earth metal salts, such as aluminum, calcium,magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes,Stearowet®, boric acid, sodium benzoate, sodium acetate, sodiumchloride, leucine, a polyethylene glycol (e.g., PEG-4000) or amethoxypolyethylene glycol such as Carbowax™, sodium oleate, sodiumbenzoate, glyceryl behenate, polyethylene glycol, magnesium or sodiumlauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sie®, a starchsuch as corn starch, silicone oil, a surfactant, and the like.

Plasticizers can be compounds used to soften the microencapsulationmaterial or film coatings to make them less brittle. Suitableplasticizers include, e.g., polyethylene glycols such as PEG 300, PEG400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propyleneglycol, oleic acid, triethyl cellulose and triacetin. Plasticizers canalso function as dispersing agents or wetting agents.

Solubilizers can include compounds such as triacetin, triethylcitrate,ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate,vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone,N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropylalcohol, cholesterol, bile salts, polyethylene glycol 200-600,glycofurol, transcutol, propylene glycol, and dimethyl isosorbide andthe like.

Stabilizers can include compounds such as any antioxidation agents,buffers, acids, preservatives and the like.

Suspending agents can include compounds such as polyvinylpyrrolidone,e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17,polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g.,the polyethylene glycol can have a molecular weight of about 300 toabout 6000, or about 3350 to about 4000, or about 7000 to about 5400,sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate,polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as,e.g., gum tragacanth and gum acacia, guar gum, xanthans, includingxanthan gum, sugars, cellulosics, such as, e.g., sodiumcarboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80,sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylatedsorbitan monolaurate, povidone and the like.

Surfactants can include compounds such as sodium lauryl sulfate, sodiumdocusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitanmonooleate, polyoxyethylene sorbitan monooleate, polysorbates,polaxomers, bile salts, glyceryl monostearate, copolymers of ethyleneoxide and propylene oxide, e.g., Pluronic® (BASF), and the like.Additional surfactants can include polyoxyethylene fatty acid glyceridesand vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil;and polyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol10, octoxynol 40. Sometimes, surfactants may be included to enhancephysical stability or for other purposes.

Viscosity enhancing agents can include, e.g., methyl cellulose, xanthangum, carboxymethyl cellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetatestearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinylalcohol, alginates, acacia, chitosans and combinations thereof.

Wetting agents can include compounds such as oleic acid, glycerylmonostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamineoleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate,sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium saltsand the like.

Injectable Formulations

Formulations suitable for intramuscular, subcutaneous, or intravenousinjection may include physiologically acceptable sterile aqueous ornon-aqueous solutions, dispersions, suspensions or emulsions, andsterile powders for reconstitution into sterile injectable solutions ordispersions. Examples of suitable aqueous and non-aqueous carriers,diluents, solvents, or vehicles include water, ethanol, polyols(propyleneglycol, polyethylene-glycol, glycerol, cremophor and thelike), suitable mixtures thereof, vegetable oils (such as olive oil) andinjectable organic esters such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case ofdispersions, and by the use of surfactants. Formulations suitable forsubcutaneous injection may also contain additives such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the growth ofmicroorganisms can be ensured by various antibacterial and antifungalagents, such as parabens, chlorobutanol, phenol, sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, such as aluminum monostearate and gelatin.

For intravenous injections, compounds described herein may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological salinebuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art. For other parenteral injections, appropriateformulations may include aqueous or nonaqueous solutions, preferablywith physiologically compatible buffers or excipients. Such excipientsare generally known in the art.

Parenteral injections may involve bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The pharmaceutical composition described herein may be ina form suitable for parenteral injection as a sterile suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Pharmaceutical formulations for parenteral administrationinclude aqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

Oral Formulations

Pharmaceutical preparations for oral use can be obtained by mixing oneor more solid excipient with one or more of the compounds describedherein, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients can include, forexample, fillers such as sugars, including lactose, sucrose, mannitol,or sorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methylcellulose, microcrystalline cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or otherssuch as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. Ifdesired, disintegrating agents may be added, such as the cross-linkedcroscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or asalt thereof such as sodium alginate.

Dragee cores can be provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Solid dosage forms may be in the form of a tablet, (including asuspension tablet, a fast-melt tablet, a bite-disintegration tablet, arapid-disintegration tablet, an effervescent tablet, or a caplet), apill, a powder (including a sterile packaged powder, a dispensablepowder, or an effervescent powder) a capsule (including both soft orhard capsules, e.g., capsules made from animal-derived gelatin orplant-derived HPMC, or “sprinkle capsules”), solid dispersion, solidsolution, bioerodible dosage form, controlled release formulations,pulsatile release dosage forms, multiparticulate dosage forms, pellets,granules, or an aerosol. In other instances, the pharmaceuticalformulation is in the form of a powder. In still other instances, thepharmaceutical formulation is in the form of a tablet, including but notlimited to, a fast-melt tablet. Additionally, pharmaceuticalformulations described herein may be administered as a single capsule orin multiple capsule dosage form. In some cases, the pharmaceuticalformulation is administered in two, or three, or four, capsules ortablets.

The pharmaceutical solid dosage forms can include a compositiondescribed herein and one or more pharmaceutically acceptable additivessuch as a compatible carrier, binder, filling agent, suspending agent,flavoring agent, sweetening agent, disintegrating agent, dispersingagent, surfactant, lubricant, colorant, diluent, solubilizer, moisteningagent, plasticizer, stabilizer, penetration enhancer, wetting agent,anti-foaming agent, antioxidant, preservative, or one or morecombination thereof. In still other aspects, using standard coatingprocedures, such as those described in Remington's PharmaceuticalSciences, 20th Edition (2000).

Suitable carriers for use in the solid dosage forms can include, but arenot limited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerine, magnesiumsilicate, sodium caseinate, soy lecithin, sodium chloride, tricalciumphosphate, dipotassium phosphate, sodium stearoyl lactylate,carrageenan, monoglyceride, diglyceride, pregelatinized starch,hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetatestearate, sucrose, microcrystalline cellulose, lactose, mannitol and thelike.

Suitable filling agents for use in the solid dosage forms can include,but are not limited to, lactose, calcium carbonate, calcium phosphate,dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose,cellulose powder, dextrose, dextrates, dextran, starches, pregelatinizedstarch, hydroxypropylmethycellulose (HPMC), hydroxypropylmethycellulosephthalate, hydroxypropylmethylcellulose acetate stearate (HPMCAS),sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride,polyethylene glycol, and the like.

Binders impart cohesiveness to solid oral dosage form formulations: forpowder filled capsule formulation, they aid in plug formation that canbe filled into soft or hard shell capsules and for tablet formulation,they ensure the tablet remaining intact after compression and helpassure blend uniformity prior to a compression or fill step. Materialssuitable for use as binders in the solid dosage forms described hereininclude, but are not limited to, carboxymethylcellulose, methylcellulose(e.g., Methocel®), hydroxypropylmethylcellulose (e.g. Hypromellose USPPharmacoat-603, hydroxypropylmethylcellulose acetate stearate (AqoateHS-LF and HS), hydroxyethylcellulose, hydroxypropylcellulose (e.g.,Klucel®), ethylcellulose (e.g., Ethocel®), and microcrystallinecellulose (e.g., Avicel®), microcrystalline dextrose, amylose, magnesiumaluminum silicate, polysaccharide acids, bentonites, gelatin,polyvinylpyrrolidone/vinyl acetate copolymer, crospovidone, povidone,starch, pregelatinized starch, tragacanth, dextrin, a sugar, such assucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol,xylitol (e.g., Xylitab®), lactose, a natural or synthetic gum such asacacia, tragacanth, ghatti gum, mucilage of isapol husks, starch,polyvinylpyrrolidone (e.g., Povidone® CL, Kollidon® CL, Polyplasdone®XL-10, and Povidone® K-12), larch arabogalactan, Veegum®, polyethyleneglycol, waxes, sodium alginate, and the like.

Suitable lubricants or glidants for use in the solid dosage forms caninclude, but are not limited to, stearic acid, calcium hydroxide, talc,corn starch, sodium stearyl fumerate, alkali-metal and alkaline earthmetal salts, such as aluminum, calcium, magnesium, zinc, stearic acid,sodium stearates, magnesium stearate, zinc stearate, waxes, Stearowet®,boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, apolyethylene glycol or a methoxypolyethylene glycol such as Carbowax™,PEG 4000, PEG 5000, PEG 6000, propylene glycol, sodium oleate, glycerylbehenate, glyceryl palmitostearate, glyceryl benzoate, magnesium orsodium lauryl sulfate, and the like.

Suitable diluents for use in the solid dosage forms can include, but arenot limited to, sugars (including lactose, sucrose, and dextrose),polysaccharides (including dextrates and maltodextrin), polyols(including mannitol, xylitol, and sorbitol), cyclodextrins and the like.

Suitable wetting agents for use in the solid dosage forms can include,for example, oleic acid, glyceryl monostearate, sorbitan monooleate,sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitanmonooleate, polyoxyethylene sorbitan monolaurate, quaternary ammoniumcompounds (e.g., Polyquat 10®), sodium oleate, sodium lauryl sulfate,magnesium stearate, sodium docusate, triacetin, vitamin E TPGS and thelike.

Suitable surfactants for use in the solid dosage forms can include, forexample, sodium lauryl sulfate, sorbitan monooleate, polyoxyethylenesorbitan monooleate, polysorbates, polaxomers, bile salts, glycerylmonostearate, copolymers of ethylene oxide and propylene oxide, e.g.,Pluronic® (BASF), and the like.

Suitable suspending agents for use in the solid dosage forms caninclude, but are not limited to, polyvinylpyrrolidone, e.g.,polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyethylene glycol, e.g., thepolyethylene glycol can have a molecular weight of about 300 to about6000, or about 3350 to about 4000, or about 7000 to about 5400, vinylpyrrolidone/vinyl acetate copolymer (S630), sodiumcarboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose,polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as,e.g., gum tragacanth and gum acacia, guar gum, xanthans, includingxanthan gum, sugars, cellulosics, such as, e.g., sodiumcarboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80,sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylatedsorbitan monolaurate, povidone and the like.

Suitable antioxidants for use in the solid dosage forms include, forexample, e.g., butylated hydroxytoluene (BHT), sodium ascorbate, andtocopherol.

Liquid formulation dosage forms for oral administration can be aqueoussuspensions selected from the group including, but not limited to,pharmaceutically acceptable aqueous oral dispersions, emulsions,solutions, elixirs, gels, and syrups. See, e.g., Singh et al.,Encyclopedia of Pharmaceutical Technology, 2^(nd) Ed., pp. 754-757(2002). In addition the liquid dosage forms may include additives, suchas: (a) disintegrating agents; (b) dispersing agents; (c) wettingagents; (d) at least one preservative, (e) viscosity enhancing agents,(f) at least one sweetening agent, and (g) at least one flavoring agent.In some embodiments, the aqueous dispersions can further include acrystalline inhibitor.

The aqueous suspensions and dispersions described herein can remain in ahomogenous state, as defined in The USP Pharmacists' Pharmacopeia (2005edition, chapter 905), for at least 4 hours. The homogeneity should bedetermined by a sampling method consistent with regard to determininghomogeneity of the entire composition. In one embodiment, an aqueoussuspension can be re-suspended into a homogenous suspension by physicalagitation lasting less than 1 minute. In another aspect, an aqueoussuspension can be re-suspended into a homogenous suspension by physicalagitation lasting less than 45 seconds. In yet another aspect, anaqueous suspension can be re-suspended into a homogenous suspension byphysical agitation lasting less than 30 seconds. In still anotherembodiment, no agitation is necessary to maintain a homogeneous aqueousdispersion.

In another aspect, dosage forms may include microencapsulatedformulations. In some embodiments, one or more other compatiblematerials are present in the microencapsulation material. Exemplarymaterials include, but are not limited to, pH modifiers, erosionfacilitators, anti-foaming agents, antioxidants, flavoring agents, andcarrier materials such as binders, suspending agents, disintegrationagents, filling agents, surfactants, solubilizers, stabilizers,lubricants, wetting agents, and diluents.

Exemplary microencapsulation materials useful for delaying the releaseof the formulations including compounds described herein, include, butare not limited to, hydroxypropyl cellulose ethers (HPC) such as Klucel®or Nisso HPC, low-substituted hydroxypropyl cellulose ethers (L-HPC),hydroxypropyl methyl cellulose ethers (HPMC) such as Seppifilm-LC,Pharmacoat®, Metolose SR, Methocer-E, Opadry YS, PrimaFlo, BenecelMP824, and Benecel MP843, methylcellulose polymers such as Methocel®-A,hydroxypropylmethylcellulose acetate stearate Aqoat (HF-LS, HF-LG,HF-MS) and Metolose®, Ethylcelluloses (EC) and mixtures thereof such asE461, Ethocel®, Aqualon®-EC, Surelease®, Polyvinyl alcohol (PVA) such asOpadry AMB, hydroxyethylcelluloses such as Natrosol®,carboxymethylcelluloses and salts of carboxymethylcelluloses (CMC) suchas Aqualon®-CMC, polyvinyl alcohol and polyethylene glycol co-polymerssuch as Kollicoat IR®, monoglycerides (Myverol), triglycerides (KLX),polyethylene glycols, modified food starch, acrylic polymers andmixtures of acrylic polymers with cellulose ethers such as Eudragit®EPO, Eudragit® L30D-55, Eudragit® FS 30D Eudragit® L100-55, Eudragit®L100, Eudragit® S100, Eudragit® RD100, Eudragit® E100, Eudragit® L12.5,Eudragit® S12.5, Eudragit® NE30D, and Eudragit® NE 40D, celluloseacetate phthalate, sepifilms such as mixtures of HPMC and stearic acid,cyclodextrins, and mixtures of these materials.

Plasticizers may include polyethylene glycols, e.g., PEG 300, PEG 400,PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propyleneglycol, oleic acid, and triacetin are incorporated into themicroencapsulation material. In other embodiments, themicroencapsulating material useful for delaying the release of thepharmaceutical compositions is from the USP or the National Formulary(NF). In yet other embodiments, the microencapsulation material isKlucel. In still other embodiments, the microencapsulation material ismethocel.

Microencapsulated compositions may be formulated by methods known by oneof ordinary skill in the art. Such known methods include, e.g., spraydrying processes, spinning disk-solvent processes, hot melt processes,spray chilling methods, fluidized bed, electrostatic deposition,centrifugal extrusion, rotational suspension separation, polymerizationat liquid-gas or solid-gas interface, pressure extrusion, or sprayingsolvent extraction bath. In addition to these, several chemicaltechniques, e.g., complex coacervation, solvent evaporation,polymer-polymer incompatibility, interfacial polymerization in liquidmedia, in situ polymerization, in-liquid drying, and desolvation inliquid media could also be used. Furthermore, other methods such asroller compaction, extrusion/spheronization, coacervation, ornanoparticle coating may also be used.

Intranasal Formulations

Intranasal formulations are known in the art and are described in, forexample, U.S. Pat. Nos. 4,476,116 and 6,391,452. Formulations thatinclude the compositions described herein, which are prepared accordingto the above described and other techniques well-known in the art areprepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, fluorocarbons, and/or other solubilizing ordispersing agents known in the art. See, for example, Ansel, H. C. etal., Pharmaceutical Dosage Forms and Drug Delivery Systems, Sixth Ed.(1995). Preferably these compositions and formulations are prepared withsuitable nontoxic pharmaceutically acceptable ingredients. Theseingredients are known to those skilled in the preparation of nasaldosage forms and some of these can be found in Remington: The Scienceand Practice of Pharmacy, 21st edition, 2005, a standard reference inthe field. The choice of suitable carriers is highly dependent upon theexact nature of the nasal dosage form desired, e.g., solutions,suspensions, ointments, or gels. Nasal dosage forms generally containlarge amounts of water in addition to the active ingredient. Minoramounts of other ingredients such as pH adjusters, emulsifiers ordispersing agents, preservatives, surfactants, gelling agents, orbuffering and other stabilizing and solubilizing agents may also bepresent. The nasal dosage form should be isotonic with nasal secretions.

For administration by inhalation described herein may be in a form as anaerosol, a mist or a powder. Pharmaceutical compositions describedherein are conveniently delivered in the form of an aerosol spraypresentation from pressurized packs or a nebulizer, with the use of asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, such as, by way of example only, gelatin foruse in an inhaler or insufflator may be formulated containing a powdermix of the compound described herein and a suitable powder base such aslactose or starch.

Therapeutic Regimens

The compositions may be administered for therapeutic applications or asa maintenance therapy, for example for a patient in remission. Thecomposition may be administered once per day, twice per day, three timesper day or more. The composition may be administered daily, every day,every alternate day, five days a week, once a week, every other week,two weeks per month, three weeks per month, once a month, twice a month,three times per month, or more. The composition may be administered forat least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months,2 years, 3 years, or more.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the compounds may be givencontinuously; alternatively, the dose of drug being administered may betemporarily reduced or temporarily suspended for a certain length oftime (i.e., a “drug holiday”). The length of the drug holiday can varybetween 2 days and 1 year, including by way of example only, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days,180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or365 days. The dose reduction during a drug holiday may be from 10%-100%,including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, can be reduced, as a function ofthe symptoms, to a level at which the improved disease, disorder orcondition is retained. Patients can, however, require intermittenttreatment on a long-term basis upon any recurrence of symptoms.

The amount of a given agent that will correspond to such an amount willvary depending upon factors such as the particular compound, theseverity of the disease, the identity (e.g., weight) of the subject orhost in need of treatment, but can nevertheless be routinely determinedin a manner known in the art according to the particular circumstancessurrounding the case, including, e.g., the specific agent beingadministered, the route of administration, and the subject or host beingtreated. The desired dose may conveniently be presented in a single doseor as divided doses administered simultaneously (or over a short periodof time) or at appropriate intervals, for example as two, three, four ormore sub-doses per day.

The pharmaceutical composition described herein may be in unit dosageforms suitable for single administration of precise dosages. In unitdosage form, the formulation is divided into unit doses containingappropriate quantities of one or more compound. The unit dosage may bein the form of a package containing discrete quantities of theformulation. Non-limiting examples are packaged tablets or capsules, andpowders in vials or ampoules. Aqueous suspension compositions can bepackaged in single-dose non-reclosable containers. Alternatively,multiple-dose reclosable containers can be used, in which case it istypical to include a preservative in the composition. By way of exampleonly, formulations for parenteral injection may be presented in unitdosage form, which include, but are not limited to ampoules, or inmulti-dose containers, with an added preservative.

The foregoing ranges are merely suggestive, as the number of variablesin regard to an individual treatment regime is large, and considerableexcursions from these recommended values are not uncommon. Such dosagesmay be altered depending on a number of variables, not limited to theactivity of the compound used, the disease or condition to be treated,the mode of administration, the requirements of the individual subject,the severity of the disease or condition being treated, and the judgmentof the practitioner.

Toxicity and therapeutic efficacy of such therapeutic regimens can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, including, but not limited to, the determinationof the LD50 (the dose lethal to 50% of the population) and the ED50 (thedose therapeutically effective in 50% of the population). The dose ratiobetween the toxic and therapeutic effects is the therapeutic index andit can be expressed as the ratio between LD50 and ED50. Compoundsexhibiting high therapeutic indices are preferred. The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosage for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED50 with minimal toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized.

According to another aspect of the invention, there is provided a methodof selecting a subject for treatment, comprising determining if thesubject has a disease induced by defective protein expression caused bythe intron retention in gene transcripts, wherein the subject isselected for treatment upon positive confirmation; and optionallytreating the subject.

The treatment may comprise correction of intron retention in the genetranscripts. The treatment may comprise hybridizing a polynucleic acidpolymer according to the invention to the gene transcript in order toinduce intron removal from the gene transcript.

According to another aspect of the invention, there is provided the useof antisense polynucleic acid polymer to normalize gene expression bycorrection of retention of introns in cancer cells.

According to another aspect of the invention, there is provided thepolynucleic acid polymer according to the invention, the compositionaccording to the invention, the vector according to the invention, orthe delivery vehicle according to the invention, for use in thetreatment or prevention of a disease.

According to another aspect of the invention, there is provided thepolynucleic acid polymer according to the invention, the compositionaccording to the invention, the vector according to the invention, orthe delivery vehicle according to the invention, for use in themanufacture of a medicament for the treatment or prevention of adisease.

The disease may be diabetes or cancer.

Where reference is made to a polynucleic acid polymer sequence, theskilled person will understand that one or more substitutions may betolerated, optionally two substitutions may be tolerated in thesequence, such that it maintains the ability to hybridize to the targetsequence, or where the substitution is in a target sequence, the abilityto be recognized as the target sequence. References to sequence identitymay be determined by BLAST sequence alignment(www.ncbi.nlm.nih.gov/BLAST/) using standard/default parameters. Forexample, the sequence may have 99% identity and still function accordingto the invention. In other embodiments, the sequence may have 98%identity and still function according to the invention. In anotherembodiment, the sequence may have 95% identity and still functionaccording to the invention.

Where reference is made to reducing or correcting intron retention, thereduction may be complete, e.g. 100%, or may be partial. The reductionmay be clinically significant. The reduction/correction may be relativeto the level of intron retention in the subject without treatment, orrelative to the amount of intron retention in a population of similarsubjects. The reduction/correction may be at least 10% less intronretentions relative to the average subject, or the subject prior totreatment. The reduction may be at least 20% less intron retentionsrelative to an average subject, or the subject prior to treatment. Thereduction may be at least 40% less intron retentions relative to anaverage subject, or the subject prior to treatment. The reduction may beat least 50% less intron retentions relative to an average subject, orthe subject prior to treatment. The reduction may be at least 60% lessintron retentions relative to an average subject, or the subject priorto treatment. The reduction may be at least 80% less intron retentionsrelative to an average subject, or the subject prior to treatment. Thereduction may be at least 90% less intron retentions relative to anaverage subject, or the subject prior to treatment.

Kits/Articles of Manufacture

Kits and articles of manufacture are provided herein for use with one ormore methods described herein. The kits can contain one or more of thepolynucleic acid polymers described herein, such as the polynucleic acidpolymers identified as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26,SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40, SEQ IDNO: 41, SEQ ID NO: 43, or SEQ ID NO: 44. The kits can also contain oneor more of the polynucleic acid polymers that are antisense topolynucleic acid polymers described herein, such as for example SEQ IDNO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ IDNO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 45,SEQ ID NO: 46, or SEQ ID NOs: 47-434. The kits can further containreagents, and buffers necessary for the makeup and delivery of thepolynucleic acid polymers.

The kits can also include a carrier, package, or container that iscompartmentalized to receive one or more containers such as vials,tubes, and the like, each of the container(s) comprising one of theseparate elements, such as the polynucleic acid polymers and reagents,to be used in a method described herein. Suitable containers include,for example, bottles, vials, syringes, and test tubes. The containerscan be formed from a variety of materials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials.Examples of pharmaceutical packaging materials include, but are notlimited to, bottles, tubes, bags, containers, bottles, and any packagingmaterial suitable for a selected formulation and intended mode ofadministration and treatment.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included.

The skilled person will understand that optional features of oneembodiment or aspect of the invention may be applicable, whereappropriate, to other embodiments or aspects of the invention.

Embodiments of the invention will now be described in more detail, byway of example only, with reference to the accompanying drawings.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1

Most eukaryotic genes contain intervening sequences or introns that mustbe accurately removed from primary transcripts to create functionalmRNAs capable of encoding proteins (1). This process modifies mRNPcomposition in a highly dynamic manner, employing interdependentinteractions of five small nuclear RNAs and a large number of proteinswith conserved but degenerate sequences in the pre-mRNA (2). Intronsplicing generally promotes mRNA accumulation and protein expressionacross species (3-5). This process can be altered by intronic mutationsor variants that may also impair coupled gene expression pathways,including transcription, mRNA export and translation. This is bestexemplified by introns in the 5′untranslated region (5′UTR) wherenatural variants or mutations modifying intron retention alter therelative abundance of transcripts with upstream open reading frames(uORFs) or other regulatory motifs and dramatically influencetranslation (6,7). However, successful sequence-specific strategies tonormalize gene expression in such situations have not been developed.

Splice-switching oligonucleotides (SSOs) are antisense reagents thatmodulate intron splicing by binding splice-site recognition orregulatory sequences and competing with cis- and trans-acting factorsfor their targets (8). They have been shown to restore aberrantsplicing, modify the relative expression of existing mRNAs or producenovel splice variants that are not normally expressed (8). Improvedstability of targeted SSO-RNA duplexes by a number of SSO modifications,such as 2′-O-methyl and 2′-O-methoxyethyl ribose, facilitated studiesexploring their therapeutic potential for a growing number of humandisease genes, including DMD in muscular dystrophy (9,10), SMN2 inspinal muscular atrophy (11), ATM in ataxia-telangiectasia (12) and BTKin X-linked agammaglobulinemia (13). Although such approaches are closeto achieving their clinical potential for a restricted number ofdiseases (8), >300 Mendelian disorders resulting from mutation-inducedaberrant splicing (14) and a growing number of complex traits may beamenable to SSO-mediated correction of gene expression.

Etiology of type 1 diabetes has a strong genetic component conferred byhuman leukocyte antigens (HLA) and a number of modifying non-HLA loci(15). The strongest modifier was identified in the proinsulin gene (INS)region on chromosome 11 (termed IDDM2) (15). Further mapping of thisarea suggested that INS is the most likely IDDM2 target (16), consistentwith a critical role of this autoantigen in pathogenesis (17). Geneticrisk to this disease at IDDM2 has been attributed to differentialsteady-state RNA levels from predisposing and protective INS haplotypes,potentially involving a minisatellite DNA sequence upstream of this gene(18,19). However, systematic examination of naturally occurring INSpolymorphisms revealed haplotype-specific proinsulin expression levelsin reporter constructs devoid of the minisatellite sequence, resultingfrom two variants in intron 1 (7), termed IVS1+5ins4 (also known asrs3842740 or INS-69) and IVS1-6A/T (rs689, INS-27 or HphI+/−) (16,20).The former variant activates a cryptic 5′ splice site of intron 1whereas adenine (A) at the latter variant, which resides 6 nucleotidesupstream of the 3′ splice site (3′ss), promotes intron retention,expanding the relative abundance of transcripts with extended 5′UTR(21). As compared to thymine (T), the A allele at IVS1-6A/T decreasesaffinity to pyrimidine-binding proteins in vitro and renders the 3′ssmore dependent on the auxiliary factor of U2 small nuclearribonucleoprotein (U2AF) (7), a heterodimer required forU2 binding,spliceosome assembly and 3′ss selection (22). Intron 1-containingtranscripts are overrepresented in IVS1-6A-derived cDNA librariesprepared from insulin producing tissues (21), are exported from thenucleus (23) and contain a short, Homininae-specific uORF thatco-evolved with relaxation of the 3′ss of intron 1 in higher primates(7). The lower proinsulin expression conferred by the A allele may leadto suboptimal presentation of proinsulin peptides in the fetal thymusand inadequate negative selection of autoreactive T cells, culminatingin autoimmune destruction of insulin-producing β cells in the pancreas(7). However, no attempts have been made to correct the low efficiencyof INS intron 1 removal from the IVS1-6A-containing pre-mRNAs and reduceintron retention to the levels observed for the disease-protective Tallele.

This study set out to search for SSOs that increase the efficiency ofINS intron 1 splicing and repress splicing silencers or decoy splicesites in the pre-mRNA to enhance proinsulin expression. SSOs reducingthe relative abundance of intron 1-retaining transcripts wereidentified, delineation of the optimized antisense target at asingle-nucleotide resolution is shown, and evidence is shown forformation of a parallel G-quadruplex adjacent to the antisense targetsequence and identification of proteins that bind to this region.

Materials and Methods Antisense Oligonucleotides

SSOs were purchased from the MWG Biotech (Germany). All SSOs andscrambled controls had a full-length phos-phorothioate backbone with2′-O-methyl ribonucleotides at the second ribose position. Apart fromINS SSOs and their scrambled versions, we employed SSOs that targetother human genes as additional controls, as described (13). Location ofeach SSO is shown in FIG. 1A and their sequences in Table 2.

Splicing Reporter Constructs

The wild-type splicing reporter carrying the type 1 diabetes associatedhaplotype termed IC was reported previously (7,21). Each constructcontains all INS exons and unabridged introns but differ in the lengthof the last exon. The IC reporters were cloned using primers D-C, D-Fand D-B; IC D-B lacks the cryptic 3′ss of intron 2. The relativeabundance of isoforms spliced to this site is lower for IC DF than forIC D-C (7,21). To test SSOs targeting the cryptic 5′ splice site ofintron 1, the IC construct was modified by a 4-nt insertion at rs3842740to create a reporter termed ICIVS1+5ins4. TSC2 and F9 constructs werereported previously (24). Plasmids were propagated in the E. coli strainDH5α and plasmid DNA was extracted using the Wizard Plus SV Miniprep kit(Promega, USA). Their inserts were completely sequenced to confirm theidentity of each of the 14 intragenic natural variants and to excludeundesired mutations.

Cell Cultures and Transfections

Human embryonic kidney 293 (HEK293), human hepatocellular livercarcinoma HepG2 and African green monkey COS7 cells were cultured inDulbecco's modified Eagle medium, 10% fetal calf serum andpenicillin/streptomycin (Life technologies, USA). Transienttransfections were carried out as described (13), using jetPRIME(Polyplus, USA) according to manufacturer's recommendations.Downregulation of U2AF35 by RNA interference (RNAi) to induce cryptic3′ss of intron 1 was performed with two hits of small interfering RNA(siRNA) U2AF35ab, as reported earlier (7,25); siRNA duplex targetingDHX36 was as described (26). The second hit was applied 24 h before theaddition of SSOs and/or reporter. Cell cultures were harvested 24 hafter addition of reporter constructs.

Analysis of Spliced Products

Total RNA was extracted with TRI Reagent and treated with DNase (Lifetechnologies, USA). The first-strand cDNA was reverse transcribed usingoligo-(dT)15 primers and Moloney murine virus reverse transcriptase(Promega, USA). Polymerase chain reaction (PCR) was carried out with acombination of a vector-specific primer PL3 and primer E targeting the3′UTR, as reported previously (7). PCR products were separated onpolyacrylamide gels and their signal intensity was measured as described(27). The identity of each mRNA isoform was confirmed by Sangernucleotide sequencing.

Circular Dichroism and Nuclear Magnetic Resonance Spectroscopy

Oligoribonucleotides for circular dichroism (CD) and nuclear magneticresonance (NMR) were purchased from Thermo Scientific, deprotectedaccording to manufacturer's instructions, lyophilized and stored at −20°C. Stock solutions were prepared from the desalted, lyophilized samplesby resuspending in milliQ water or KCl buffer (100 mM KCl,10mMK₂HPO4/KH2PO₄, pH 7.0, milliQ water) to a final concentration of 2-4μM. CDspectra were acquired using a PiStar-180 spectrophotometer(Applied Photophysics Ltd, Surrey, UK), equipped with a LTD6Gcirculating water bath (Grant Instruments, UK) and thermoelectrictemperature controller (Melcor, USA). Samples were heated in the cell to95° C. for a total period of 15 min, samples were then annealed byallowing to cool to room temperature for a minimum period of 4 h. CDspectra were recorded over a wavelength range of 215-340 nm using a 1 cmpath length strain-free quartz cuvette and at the temperaturesindicated. Data points recorded at 1 nm intervals. A bandwidth of 3 nmwas used and 5000 counts acquired at each point with adaptive samplingenabled. Each trace is shown as the mean of three scans (±SD). CDtemperature ramps were acquired at 265 nm corresponding to the bandmaxima of the folded quadruplex species. Ranges between 5 and 99° C.were used, with points acquired at 0.5° C. intervals with a 120-180 stime step between 0.5° C. increments. Points were acquired with 10,000counts and adaptive sampling enabled. Heating and cooling studies werecompared to check for hysteresis and overall reversibility. NMR spectra(1H) were collected at 800 MHz using a Bruker Avance III spectrometerwith a triple resonance cryoprobe. Standard Bruker acquisitionparameters were used. Data were collected using Topspin (v. 3.0) andprocessed in CCPN Analysis (v. 2.1).

Pull-Down Assays and Western Blotting

In vitro transcription was carried out using MEGAshortscript™ T7(LifeTechnologies, USA) and T7-tagged PCR products amplified withprimers 5′-ATTAATACGACTCACTATAGGGCTCAGGGTTCCAGG (SEQ ID NO: 463) and5′-TGCAGCAGGGAGGACG (SEQ ID NO: 464), and DNA of the indicated plasmidsas a template. Indicated synthetic RNAs were purchased from Eurofins UK.Five hundred pmols of each RNA was treated with 5 mM sodium m-periodateand bound to adipic acid dehydrazide agarose beads (Sigma, USA). Beadswith bound RNAs were washed three times in 2 ml of 2 M NaCl and threetimes in buffer D (20 mM HEPES-KOH, pH 7, 6.5% v/v glycerol, 100 m MKCl, 0.2 mM EDTA, 0.5 mM dithiothreitol), incubated with HeLa nuclearextracts and buffer D with heparin at a final concentration of 0.5mg/ml. Unbound proteins were washed five times with buffer D. Boundproteins were separated on 10% sodium dodecyl sulphate polyacrylamidegel electrophoresis, stained by Coomassie blue and/or blottednitrocellulose membranes.

Western blotting was carried out as described (7). Antibodies werepurchased from Sigma (hnRNP E1/E2, product number R4155, U2AF65, productnumber U4758 and SFRS2, product number S2320), Abcam (DHX36, productnumber ab70269) and Millipore (SC35, clone 1SC-4F11). Antiserum againsthnRNP F and hnRNP H provided by Prof. Douglas Black, UCLA.

Mass Spectrometry Analysis

Following trypsin digestion, samples were freeze dried and resuspendedwith 25 μl of 5% ACN/0.1% formic acid for mass spectrometry (MS).Peptides were analysed by LC/MS/MS using a Surveyor LC system and LCQDeca XP Plus (ThermoScientific). The raw data files were converted intomascot generic files using the MassMatrix File Conversion Tool (Version2.0; http://www.massmatrix.net) for input into the Mascot searchingalgorithm (Matrix Science).

Enzymatic Structural Probing

RNA secondary structure determination with the use of limited V1 RNAse(Ambion), T1 RNAse (Ambion) and S1 nuclease (Fermentas) digestion hasbeen described in detail elsewhere (28). Briefly, 1 μg aliquots of RNAsfrom the insertion (ins) and deletion (del) pre-mRNAs were digested with0.002 U of RNAse V1, 0.05 U of RNAse T1 and 19 U of S1 nuclease in a 100μl at 30.0 for 10 min. An enzyme free aliquot was used as a control (C).The cleaved RNAs were retro-transcribed according to standard protocolsusing antisense primers labeled with [³²P]-ATP at the 5′ end.

Results

Antisense Oligonucleotides that Promote Pre-mRNA Splicing of a WeakIntron in 5′UTR

To identify SSOs capable of reducing retention of INS intron 1 andincrease splicing-mediated translational enhancement, we designed aseries of 2′-O-methyl-modified phosphorothioate SSOs, individuallyco-expressed each SSO with a splicing reporter construct carryinghaplotype IC in HEK293 cells and examined the relative abundance ofexogenous mRNA products (FIGS. 1A and B). The IC haplotype in thereporter was devoid of the minisatellite sequence and contained a totalof 14 polymorphic sites (7,20), including the A allele at rs689. Thisallele inhibits intron 1 splicing and yields lower proinsulin levels ascompared to the more common T allele (21). SSOs targeting intron 1 andexon 2 were chosen in regions that showed the most prominent alterationsof exon inclusion or intron retention in previous systematic deletionanalyses of these sequences (7). SSOs in exon 3 were located betweenauthentic 3′ss of intron 2 and a strong competing cryptic 3′ss 126 ntdownstream to identify pre-mRNA motifs that modify their usage (FIG.1A). Of the initial set of 15 INS SSOs tested in HEK293 cells, 11 showedreproducible alterations in the relative abundance of mRNA isoforms(Table 2). Intron 1 retention was significantly reduced by a singleoligoribonucleotide SSO21 (P<0.01, Mann-Whitney rank sum test; FIG. 2A).SSO21 targeted intron 1 positions 59-74, encompassing a motif (termedde15) previously found to confer the largest reduction of intronretention upon deletion (7). The decrease in intron retention levelsinduced by SSO21 was dose-dependent (FIG. 2A) and was also observed inHepG2 cells and Chlorocebus aethiops COST cells, consistent withubiquitous expression and a high degree of evolutionary conservation ofspliceosome components that employ auxiliary splicing sequences (1,2).In addition to reducing intron 1 retention, SSO21 promoted cryptic 3′ssof intron 2 (FIG. 2A). However, this effect was also seen for other INSSSOs and for scrambled controls (FIG. 3 and Table 2), suggestingnon-specific interactions. To confirm that the SSO21-induced enhancementof intron 1 splicing is not facilitated by the cryptic 3′ss of intron 2,we co-transfected this SSO with a shorter reporter lacking this site andretaining only the first 89 nucleotides of exon 3. FIG. 2B shows thatSSO21 was capable of promoting intron 1 splicing to the same extent asthe reporter with longer exon 3. In contrast, the SSO21-induced decreaseof intron retention was not observed for the reporter lacking the del15segment. Apart from intron retention, an increase of exon 2 skipping wasobserved for five SSOs, including SSO8 that bound downstream of thecryptic 3′ss of intron 1 (cr3′ss+81; FIGS. 1 and 3C, Table 2). Thiscryptic 3′ss was induced by RNAi-mediated depletion of the small subunitof U2AF (U2AF35) and was not reversed by a bridging oligoribonucleotide(SSO4) in cells lacking U2AF35; instead exon 2 skipping was observed(FIG. 3C). Depletion of U2AF35 also repressed the cryptic 3′ss of intron2. Taken together, a single SSO was identified that reduced INS intron 1retention in several primate cell lines.

Optimization of the Intron Retention Target at the Single NucleotideLevel

Interestingly, other SSOs designed to target the del5 segment did notreduce intron 1 retention, except for a small effect of SSO20 (FIGS. 1Aand 2A). To test the importance of nucleotides flanking SSO21 and to mapthe optimal target at a single-base resolution, a detailed antisensemicrowalk was carried out in this region. The INS reporter wasindividually co-transfected with additional eighteen 16-mers bound 1-9nucleotides 5′ and 3′ of SSO21 into HEK293 cells and their RNA productsexamined. Intron 1 retention was most repressed by SSO21 and by SSOsthat were shifted by 1-2 nucleotides in each direction (FIG. 4). Inagreement with the initial screen, SSOs targeting more than one cytosinein the upstream run of four Cs (C4, see SSO1 and SSO2, FIG. 1A) were noteffective (SSO21-3r through SSO21-10r, FIG. 4). In the oppositedirection, SSOs targeting consecutive Gs, which are often found inintronic splicing enhancers (29-31), increased intron retention. Thus,the optimal antisense target for reducing retention of INS intron 1 wasmapped at a single nucleotide resolution to a region previouslyidentified as the most repressive by a systematic deletion analysis ofthe entire intron (7).

Antisense Target for Intron Retention is Adjacent to a Parallel RNAQuadruplex

It was noticed that the target was sandwiched between two intronicsegments predicted to form stable RNA guanine (G) quadruplexes (intron 1nucleotides 36-61 and 78-93; highlighted in FIG. 4A). These structuresare produced by stacking G-quartets that consist of four Gs organized ina cyclic Hoogsteen hydrogen bonding arrangement (32) and have beenimplicated in important cellular processes, including replication,recombination, transcription, translation (33,34) and RNA processing(35-39). To test if they are formed in vitro, synthetic ribonucleotidesderived from this region were employed in CD spectroscopy that has beenused widely to characterize DNA and RNA quadruplex structures in vitro(40-43). The CD spectrum of a downstream 19-mer (termed CD1) recordedbetween 215 and 330 nm at 25° C. revealed strong positive ellipticity at265 nm with negative intensity at around 240 nm, indicative of aparallel quadruplex (FIG. 5A). To confirm the presence of a quadruplex,rather than other stable secondary structure motifs, UV absorbancespectra was recorded at 5° C. and 95° C. The UV absorbance differencespectrum at the two temperatures (below and above the melting transitionpoint) showed the characteristic hyperchromic shift at ˜295 nm and adouble maximum at 240 nm and 280 nm, providing evidence for formation ofa stable parallel-stranded RNA quadruplex in vitro. This was confirmedby 1H NMR studies of CD1 (FIG. 5B) which showed a characteristicenvelope of signals between 10 and 12 ppm corresponding to HoogsteenH-bonded Gs within G-tetrad structures. Thermal stability measurementsby CD produced a highly reversible sigmoidal co-operative unfoldingtransition with a T_(m)=56.8±0.2° C. (FIG. 5C). FIG. 5D (upper panel)shows a possible arrangement of the 19-mer into two stacked G-tetradsconnected by relatively short loop sequences of 1-4 nucleotides.

Conformational Transition Model for Splicing Inhibitory Sequences in INSIntron 1

CD of a synthetic 20-mer derived from a region upstream of the antisensetarget (termed CD2) also showed evidence of stable structure formation,giving a broader absorption envelope centered around 270 nm and asigmoidal thermal unfolding transition (T_(m)=69.0±0.45° C.; FIG. 5A).Unlike the downstream oligo CD1, no hyperchromic shift in the UV wasfound in the thermal difference spectrum. However, a well-defined set ofsharp signals in the 1H NMR spectrum between 12 and 14 ppm that differedfrom those for CD1 showed the formation of Watson-Crick H-bonded basepairs characteristic of double-stranded RNA (FIG. 5B). Secondarystructure predictions of overlapping intronic segments using Mfoldsuggested that the pre-mRNA forms stable local stem-loops; one of themwas further stabilized by a G→C mutation (termed G2; FIG. 5D, lowerpanel) that increased intron 1 retention (7). Another G→C substitution(termed G3) located further downstream and destabilizing the quadruplexstructure (FIG. 5D, upper panel) also repressed intron splicing (7).Finally, CD2 oligonucleotides containing either A or G at asingle-nucleotide polymorphism (FIG. 4A and (20) exhibited very similarCD spectra with well-defined melting transitions and T_(m) values,suggesting that the G and A alleles form the same structure.

To test further the importance of a tentative equilibrium betweencanonical and noncanonical structures in intron splicing, a combinationof CD, NMR and mutagenesis experiments was used (FIG. 6). Anoligoribonucleotide CD3 encompassing the 5′ end of the intron retentiontarget was synthesized and stem-loops/quadruplex were predicted (FIGS.4A and 6A). A mutated version CD4 was also synthesized, which carriedtwo C→U transitions destabilizing the hairpin but maintaining stabilityof the quadruplex. The same mutation was also introduced in the ICreporter construct transfected into HEK293 cells. The NMR spectrum ofCD3 revealed the co-existence of signals for both G-tetrad and canonicalbase-paired hairpin structures (termed H1 and H2) in equilibrium (FIGS.6B and C). The effects of Mg2+ on the conformational equilibrium betweenquadruplex and hairpin were investigated by adding 2 mM and then 6 mMMgCl₂ to the buffered solution containing 100 mM KCl. As reported byBugaut et al. (57), the conformational equilibrium was not significantlyperturbed by the addition of Mg2+ in the presence of KCl. Thus, weobserved formation of the RNA hairpin and quadruplex structures in anenvironment that mimics the cellular context where both K⁺ and Mg²⁺ ionswere present at high concentrations. The CD melting curve showed a broadtransition (T_(m)=79.9° C.), consistent with multiple conformationalstates with different stabilities. The CC→UU mutation in CD4 resulted inthe loss of NMR signals for H1 (FIG. 6B) and a reduction in the Tm by13° C., consistent with the selective destabilization of the more stablehairpin H1, leading to an increase in the population of H2 inequilibrium with the quadruplex. Transient transfections showed that theCC→UU mutation improved intron 1 splicing while a mutation termed M1predicted to destabilize both the quadruplex and the hairpin had only asmall effect (FIG. 6D, Table 1A).

To explore how the equilibrium of these structures affects intronsplicing more systematically, a series of mutated constructs wereprepared in order to destabilize/maintain predicted quadruplex, H1/H2structures and two cytosine runs (Table 1A). Their transcripts showedsignificant differences in intron retention levels (FIG. 7; P=0.0001,Kruskal-Wallis one-way ANOVA on ranks). First, elimination of theG-quadruplex increased intron 1 retention, which was further enhanced byremoving each cytosine run (cf. mutations 4-6 with the wild-type,P=0.0004). These mutations appeared to have additive effects on intronretention (cf. wild-type versus mutations 1 or 9; 3 versus 2 and 4versus 5). Second, the increased intron retention in the absence of theG-quadruplex was not altered by removing H1 and H2, but theirelimination enhanced exon skipping (cf. isoform 2 for mutations 4 versus6). Third, when only one of the two C4 runs was present, removal of H1somewhat improved intron 1 splicing (cf. 8 versus 9), consistent with astatistically significant correlation between intron retention andpredicted stability of tested RNAs (FIG. 7B). The efficiency of intronsplicing was thus controlled by conformational transitions betweencanonical and noncanonical structures in equilibrium.

TABLE 1AINS intron 1 mutations altering predicted RNA G quadruplexes, stem loops andtwo cytosines runs in plasmid constructs Free Input sequence Predictedenergy for computation RNA C The most stable RNA (kcal/ Mutationpredictions1 quadruplex H2 H1 runs structure mmol) Wildtype sequenceGGAUUCCAGGG UGGUGGACCC CAGGCCCC + + + +

−9.8 Del5 GGAUUCCAGGG UGGCUGG---- -------- + + − −

−2.7 M1 GGAUUCCAGGG UGGUGCACGC CAGGCCCC + − − −

−9.3 1 GGAUUCCAGGG UGGUGGACCC GAGGCGCC + + + −

−8.4 2 GGAUUCCAGGA UGGUAGGACCC GAGGCGCC + − − −

−2.8 3 GGAUUCCAGGA UGGUAGGACCC CAGGCCCC + − − +

−4.3 4 GGAUUCCAGGG UUGCUGGACCC CAGUCCCC − + + +

−11.1 5 GGAUUCCAGGG UUGCUGGACCC GAGUCGCC − + + −

−11.3

Protein-RNA Interactions in the Region Targeted by Winner SSOs

To identify proteins that interact with RNAs encompassing the antisensetarget and/or associated canonical and noncanonical structures,pull-down assays were carried out using wild type and del5 RNAstranscribed from T7-tagged PCR products, a synthetic RNA (CD5)representing the target sequence, and a control oligo containing a 3′ ssCAG, termed AV3. Western blotting showed that both wild type and del5transcripts bound hnRNPs F/H but this binding was absent for CD5 (FIG.7C). These proteins were also detected by MS/MS analysis ofdifferentially stained fragments from pull down gels with wild type anddel5 RNAs as compared to beads-only controls. Two antibodies againstSRSF2, which showed the highest score for putative binding activityamong several SR proteins, failed to detect any specific interaction(FIG. 7C). Although the signal from hnRNP E1/E2, which constitute amajor poly(C) binding activity in mammalian cells (44), was abovebackground for del5 (FIG. 7C), no change in intron retention wasobserved in cells lacking hnRNP E1/E2.

Splicing Pattern of G-Rich and G-Poor Reporters Upon DHX36 Depletion

RNA G-quadruplexes bind helicase DHX36, which is capable of convertingquadruplex RNA to a stable duplex and is a major source ofquadruplex-resolving activity in HeLa cells (26,45). DHX36 wascrosslinked to an intronic splicing enhancer in the ATM pre-mRNA (46)and could unwind the quadruplex structure within the 5′ region of TERC(26). To test if DHX36 depletion can influence INS splicing,G-quadruplex-poor and -rich reporters were transiently transfected (FIG.8A, Table 1) into depleted cells. Control constructs were chosen to giveapproximately equal representation of spliced products, which wasachieved by weakening the branch site (24), thus providing a sensitiveex vivo splicing assay. However, despite efficient DHX36 depletion (FIG.8B), statistically significant alterations of INS intron 1 retentionwere not seen in either short or long constructs, nor did we observemajor changes in G-poor and G-rich controls (FIG. 8C-E). These resultsare in agreement with a previous lack of significant enrichment ofquadruplex sequences among transcripts down regulated in DHX36-depletedcells (47) and with the absence of ATM response to the knockdown (46).

SSO-Induced Repression of a Population-Specific Cryptic 5′ Splice Siteof INS Intron 1

In addition to rs689, INS intron 1 splicing is influenced by apolymorphic TTGC insertion at rs3842740 located in the vicinity of thenatural 5′ss (21). This insertion is present in a quarter of all Africanchromosomes but is absent on Caucasian IC haplotypes (20). The insertionactivates a downstream cryptic 5′ss (FIG. 1A), extending the 5′UTR ofthe resulting mRNAs by further 26 nucleotides and repressing proinsulinexpression (7,21). To test if the new 5′ss can be efficiently inhibitedby SSOs, the same insertion was introduced in the IC construct and thewild type and mutated reporters co-expressed with a bridgingoligoribonucleotide termed SSO10. Although the cryptic splicing wasinhibited, canonical splicing of intron 1 was not completely restoredeven at high SSO10 concentrations, most likely as a result of suboptimalrecognition of the authentic 5′ss weakened by the insertion.

To gain initial insights into folding of 5′UTR sequences in the presenceand absence of the insertion, enzymatic structural probing was carriedout using partial RNA digestion with single- and double-strand specificRNAses. The overall cleavage positions and intensities detected for thewild-type RNA were broadly consistent with mfold predictions, in whichtwo major stem loop regions (SL1 and SL2) were interrupted by severalinternal bulges. Both the structural probing and mfold predictionssuggested that the insertion at rs3842740 extended the central bulge inSL1 as the number of T1 and S1 cleavages in this region increased incontrast to the remaining portions of SL1 and in SL2. Finally,transcripts were not digested by RNase V1 in regions showing quadruplexformation in vitro.

Discussion Antisense Intron Retention Target in a Splicing Silencer ofINS Intron 1

Here it is demonstrated the first use of antisense technology to reduceretention of the entire intron in mature transcripts and to modify thehaplotype-dependent INS expression using SSOs. Identification of winnerSSOs that compensate the adverse impact of the A allele at rs689 onefficient RNA processing was facilitated by systematic mutagenesis ofintron 1 (7), and by macro-(FIG. 1) and micro-walk (FIG. 4) strategies.Interestingly, the target sequence contains a tandem CAG (G/C) motif,which resembles a 3′ss consensus (FIG. 4). Such ‘pseudo-acceptors’ werepreviously implicated in splice-site repression experimentally (27) andare overrepresented in splicing silencers. For example, the twotetramers are more common among high-confidence 102 intronic splicingsilencers (49) and are depleted in 109 enhancers (50) identified byfluorescence activated screen of random 10-mers. The YAG motifs werealso more frequent than expected among QUEPASA splicing silencers (51),suggesting that they are important functional components of theretention target. The intervening cytosine tract may also play animportant role as the frequency of C4 runs among QUEPASA silencers is ˜2times higher than expected. These motifs were also found in 4% ofintronic splicing regulatory elements identified by a systematicscreening of sequences inserted at positions −62/−51 relative to atested 3′ss (52). This study identified an element termed ISS22(AAATAGAGGCCCCAG; SEQ ID NO: 465) that shared a 3′ nonamer (underlined)with the optimal intron retention target. However, unlike an optimal3′ss recognition sequence of AV3, our pull-down assay coupled withwestern blotting revealed only a very weak binding if any to U2AF65(FIG. 7C).

Conformational Transition Between Quadruplex and Hairpins in RNAProcessing Control

The antisense target was identified just upstream of a potentialG-quadruplex forming RNA whose structure was subsequently confirmed byCD and NMR analysis (FIGS. 1A and 5). RNA quadruplexes are more stablethan their DNA counterparts, have been increasingly implicated inregulation of RNA metabolism (33,34,41,42) and offer unique avenues fordrug development (53). The 2-quartet quadruplexes are thermodynamicallyless stable than their 3- or 4-quartet counterparts and are probablykinetically more labile, yet they still display pronounced stability andmay serve as more compliant and dynamic switches between quadruplex andnon-quadruplex structures in response to cellular environment (54-56).The winner SSOs may block interactions with trans-acting factors, alterhigher-order structures, the rate of RNA-protein complex formation orimpair conformational transition between the 2-quartet quadruplex andH1/H2 (FIG. 5). A similar transition has been recently described for aquadruplex not predicted ab initio (57), raising a possibility thatadditional sequences in the G-rich intron 1 may participate in theequilibria near the antisense target, possibly involving multiplequadruplex motifs and competing stem-loops.

The binding (FIG. 7C) and functional experiments showing the increasedintron 1 retention upon hnRNP F/H depletion and the opposite effect uponhnRNP F/H overexpression (7) indicate that these proteins interact withkey splicing auxiliary sequences in this intron. In contrast to aprevious report concluding that hnRNP F binds directly to the RNAquadruplex (58), hnRNP F has been shown to prevent formation of RNAquadruplexes by binding exclusively single-stranded G-tracts (59).Predictions based on primate genomes suggest that the majority ofputative quadruplexes are likely to fold into canonical structures (60).Decreased pre-mRNA occupancy by these proteins, presumably promotingquadruplex formation (59), and potentially reducing splicing efficiency.[HD1]

RNA Quadruplexes in Coupled Splicing and Translational Gene ExpressionControl

RNA quadruplexes were predicted in ˜8.0% of 5′UTR and were proposed toact as general inhibitors of translation (60,61). INS intron 1 is weaklyspliced and U2AF35-dependent (7) and a significant fraction of intron1-containing transcripts is exported from the nucleus (23). Thissuggests that the RNA G-quadruplex formed by CD1 could influencetranslation of these mRNAs, which contain a three-amino acid uORFspecific for Homininae (7). This uORF markedly inhibits proinsulinexpression and is located just a few base-pairs downstream, prompting aconcept that the G-quadruplexes can promote translation by sequesteringuORFs. Functional 2-quartet quadruplexes are required for activity ofinternal ribosomal entry sites (54).

TABLE 1 Density of predicted RNA G-quadruplexes in reporter constructsReporter TSC2 F9 INS G-quadruplexes per 0.25 0.05 0.27 nucleotide^(a) Gscore per 0.20 0.04 0.22 nucleotide^(a) ^(a)The length ofnon-overlapping quadruplex sequences and their G scores were computed asdescribed (78).

Antisense Strategies for Dependencies in Splice-Site Selection

Apart from canonical mRNA isoform 4, isoforms 2, 3 and 6 (FIG. 1B) havebeen found in expressed sequence tag databases derived from cDNAlibraries from insulin producing tissues (21). This suggests thatcryptic splice sites produced by the reporter construct are recognizedin vivo and that our haplotype-dependent reporter system recapitulatesthese events accurately in cultured cells no matter whether the cellsexpress or not endogenous insulin. Apart from repressing intron1-retaining transcripts, optimal SSOs increased utilization of cryptic3′ss of exon 3 (FIG. 2). This undesired effect could be explained bycoordination of splicing of adjacent exons and introns, which wasobserved previously for individual genes and globally (63-67). Also,G-richness downstream transcription start sites have been associatedwith RNA polymerase II pausing sites (68). Although the two robustlycompeting 3′ss of intron 2 are likely to respond to non-specific signalsthat influence RNA folding (FIG. 3, Table 2), it might be possible toalleviate the observed dependencies and reduce cryptic 3′ss activationusing SSO combinations at linked splice sites and examine theirsynergisms or antagonisms, benefiting from the use of full-geneconstructs as opposed to minigenes.

Multifunctional Antisense Oligonucleotides to Reduce INS Intron 1Retention

Since the first use of 2′-O-methyl-phosphorothioate SSOs (69), this typeof chemical modification has been successfully exploited for many invitro and in vivo applications (9,10,70). To further fine-tuneexpression of mRNA isoforms, optimized SSOs can be designed to tethersuitable trans-acting splicing factors to their target sequences(11,71). An obvious candidate for this system is U2AF35 because intron 1is weak as a result of relaxation of the 3′ss in higher primates and isfurther undermined by the A allele at rs689, which renders this intronhighly U2AF35-dependent (FIG. 3) (7). Apart from U2AF35, future bi- ormultifunctional antisense strategies can employ binding platforms forsplicing factors previously shown to influence INS intron 1 and exon 2splicing, such as Tra2β or SRSF3 (7). Tra2β is likely to bind the SSO6target which forms a predicted stable hairpin structure with a potentGAA splicing enhancer in a terminal loop (FIG. 3B). SRSF3 is requiredfor repression of the cryptic 3′ss of intron 2 (7) and bindspyrimidine-rich sequence with a consensus (A/U)C(A/U)(A/U)C (72). TheCAUC motif, which interacts with the RNA-recognition motif of SRSF3(73), is present just upstream of the cryptic 3′ss.

Normalizing Intron Retention Levels in Human Genetic Disease

These results provide an opportunity to use non-genetic means tocompensate less efficient splicing and lower INS expression fromhaplotypes predisposing to type 1 diabetes.

Common variants such as rs689 contribute to a great extent to theheritability of complex traits, including autoimmune diseases (74), buttheir functional and structural consequences are largely unknown. Ifoptimized INS SSOs can be safely and efficiently introduced into thedeveloping thymus, this approach may offer a novel preventive approachto promote tolerance to the principal self-antigen in type 1 diabetes.The most obvious candidates for such intervention are mothers who had anaffected child homozygous for disease-predisposing alleles at both HLAand INS loci. Such genotypes were associated with an extremely highdisease risk for siblings (75). Apart from primary prevention of type 1diabetes, future SSO-based therapeutics might be applicable to patientswith significant residual β-cell activity at diagnosis and to those whoare eligible to receive β-cell transplants and may benefit fromincreased intron-mediated enhancement of proinsulin expression fromtransplanted cells. It is also possible to envisage use of thistherapeutic modality for other patients with diabetes through a moredramatic enhancement of intron splicing and proinsulin expression bytargeting multiple splicing regulatory motifs with multifunctional SSOs.The SSOs may have utility in thymic epithelial cells and 13-cells thatmay provide a more natural system for testing their impact on both exo-and endogenous proinsulin expression. Finally, similar antisensestrategies may help reduce pervasive intron retention in cancer cellsresulting from somatic mutations of splicing factor genes, asillustrated by specific substitutions in the zinc finger domain ofU2AF35 in myeloproliferative diseases (76).

Reducing Intron Retention in Malignant Cells

A set of 146 intronic sequences that are preferentially retained inU2AF-deficient HEK293 cells was selected using RNAseq data fromreplicated, polyA-selected samples, followed by inspection of eachintron retention event in genome browsers. These sequences wererepeat-masked using a sensitive version of RepeatMasker, available athttp://www.repeatmasker.org/cgi-bin/WEBRepeatMasker. Because the optimalantisense target for reducing INS intron 1 retention [Kralovicova J etal (2014). Nucleic Acids Res doi: 10.1093/nar/gku507, published on 17Jun. 2014] overlapped intronic splicing regulatory elements conserved inmammals [Yeo G W, et al (2007). PLoS Genet 3:e85], including CCCAG,AGGCC (FIG. 4 in Kralovicova et la. 2014), antisense targets in intronicsegments containing these short penta- to heptamer motifs and anindependently derived set of intronic splicing regulatory motifs[Voelker R B, & Berglund J A (2007). Genome Res 17:1023-1033], wereselected, thus increasing the chance of oligonucleotides interactingwith these targets to influence RNA processing. The target sequenceswere subjected to routine antisense oligonucleotides design, includingremoval of sequences containing C runs to avoid G-quadruplex formation.The proximity of both 5′ and 3′ splice sites, polypyrimidine tracts,branch sites and suprabranch regions were also avoided. This selectionyielded a set of 388 compounds (Table 3), covering ˜15% of the totallengths of U2AF-sensitive introns and representing ˜0.001% of all humanintronic sequences. Thus, this set of oligonucleotides target regionsenriched for splicing inhibitory sequences of U2-dependent introns,which do not have sufficient help from auxiliary factors in malignantcells that sustain mutations or deletions in the U2 pathway.

Insulin Related Sequences

Candidate SSO sequences CD5 RNA form-CUGCAGAGCUGGGGCCUG (SEQ ID NO: 1)DNA form-CTGCAGAGCTGGGGCCTG (SEQ ID NO: 2)Binds to caggccccagcucugcag (SEQ ID NO: 3) SSO21*RNA form-UGCAGAGCUGGGGCCU (SEQ ID NO: 4)DNA form-TGCAGAGCTGGGGCCT (SEQ ID NO: 5)Binds to aggccccagcucugca (SEQ ID NO: 6) SSO21-2r*RNA form-GCAGAGCUGGGGCCUG (SEQ ID NO: 7)DNA form-GCAGAGCTGGGGCCTG (SEQ ID NO: 8)Binds to caggccccagcucugc (SEQ ID NO: 9) SSO21-3rRNA form-CAGAGCUGGGGCCUGG (SEQ ID NO: 10)DNA form-CAGAGCTGGGGCCTGG (SEQ ID NO: 11)Binds to ccaggccccagcucug (SEQ ID NO: 12) SSO21-4rRNA form-AGAGCUGGGGCCUGGG (SEQ ID NO: 13)DNA form-AGAGCTGGGGCCTGGG (SEQ ID NO: 14)Binds to cccaggccccagcucu (SEQ ID NO: 15) SSO21-5rRNA form-GAGCUGGGGCCUGGGG (SEQ ID NO: 16)DNA form-GAGCTGGGGCCTGGGG (SEQ ID NO: 17)Binds to ccccaggccccagcuc (SEQ ID NO: 18) SSO21-6rRNA form-AGCUGGGGCCUGGGGU (SEQ ID NO: 19)DNA form-AGCTGGGGCCTGGGGT (SEQ ID NO: 20)Binds to accccaggccccagcu (SEQ ID NO: 21) SSO21-7rRNA form-GCUGGGGCCUGGGGUC (SEQ ID NO: 22)DNA form-GCTGGGGCCTGGGGTC (SEQ ID NO: 23)Binds to gaccccaggccccagc (SEQ ID NO: 24) SSO21-8rRNA form-CUGGGGCCUGGGGUCC (SEQ ID NO: 25)DNA form-CTGGGGCCTGGGGTCC (SEQ ID NO: 26)Binds to ggaccccaggccccag (SEQ ID NO: 27) SSO21-9rRNA form-UGGGGCCUGGGGUCCA (SEQ ID NO: 28)DNA form-TGGGGCCTGGGGTCCA (SEQ ID NO: 29)Binds to uggaccccaggcccca (SEQ ID NO: 30) SSO21-10rRNA form-GGGGCCUGGGGUCCAG (SEQ ID NO: 31)DNA form-GGGGCCTGGGGTCCAG (SEQ ID NO: 32)Binds to cuggaccccaggcccc (SEQ ID NO: 33) SSO21-14PRNA form-CUGCAGAGCUGGGGCC (SEQ ID NO: 34)DNA form-CTGCAGAGCTGGGGCC (SEQ ID NO: 35)Binds to ggccccagcucugcag (SEQ ID NO: 36) SSO21-15fRNA form-GCUGCAGAGCUGGGGC (SEQ ID NO: 37)DNA form-GCTGCAGAGCTGGGGC (SEQ ID NO: 38)Binds to gccccagcucugcagc (SEQ ID NO: 39) SSO21-16fRNA form-UGCUGCAGAGCUGGGG (SEQ ID NO: 40)DNA form-TGCTGCAGAGCTGGGG (SEQ ID NO: 41)Binds to ccccagcucugcagca (SEQ ID NO: 42) SSO21-17fRNA form-CUGCUGCAGAGCUGGG (SEQ ID NO: 43)DNA form-CTGCTGCAGAGCTGGG (SEQ ID NO: 44)Binds to cccagcucugcagcag (SEQ ID NO: 45)Sequence of target region of pre-mRNA transcript (e.g. for binding SSOs)  cuggaccccaggccccagcucugcagcag (SEQ ID NO: 46)Note: candidates marked with ″*″ are the winner oligos discussed in FIG.4.

TABLE 2 Effects on the relative abundance of INS SSO Location1Sequence (5′-3′) mRNA isoforms  1 Intron 1 (del5, del6) AGCUGGGGCCUGGGGUActivation of the cryptic 3′ ss of intron 2  2 Intron 1 (del5, del6)UGCAGAGCUGGGGCCUGG Activation of the cryptic GGU 3′ ss of intron 2  3Intron 1 (del8, del9) CAUGCUUCACGAGCCCAG Increased exon 2 CC skipping  4Exon 2 (cryptic 3′ ss AAGGCUGCGGCUGGGUC Increased exon 2 +81, skippingdel8, del9)  5 Exon 3 UGGUAGAGGGAGCAGAUG Decreased efficiency of UGintron 2 splicing; Activation of the cryptic 3′ ss of intron 2  6 Exon 3UGGUACAGCAUUGUUCCA Activation of the cryptic CA 3′ss of intron 2 at high concentration  8 Exon 2 (del13-15)CGCACACUAGGUAGAGAG Increased exon 2 C skipping  9 Exon 1GAUGCAGCCUGUCCUGGA None G 10 Intron 1 (del1, del2, GAGCCCACCUGACGCAAAPartial restoration of cryptic 5′ splice site  GGC authentic 5′splice site +30) 16 Exon 1 UGGAGGGCUGAGGGCUGC None U 17 Exon 1AUGGCCUCUUCUGAUGCA None 18 Intron 1 (del9, del10) UCACCCCCACAUGCUUCIncreased exon 2 skipping 19 Intron 1 (del9) ACAUGCUUCACGAGIncreased exon 2 skipping 20 Intron 1 (del5) CUGGGGCCUGGGGUMinor reduction of intron 1 retention; activation of the cryptic 3′ss of intron 2 21 Intron 1 (del5, del6) UGCAGAGCUGGGGCCUReduction of intron 1 retention; activation of the cryptic 3′ss of intron 2 1sc Scrambled control AGGUGCUCGCGGGUGG None 2scScrambled control GGGUGGAAGCGUCCGGUC Stimulation of the cryptic GUG 3′ss of intron 2 3sc Scrambled control ACACACUGUGCCUCGCCA None GC 6scScrambled control GACUCACUUGCCGUAGUU Stimulation of the cryptic AA 3′ss of intron 2 8sc Scrambled control CACGCUCAGUAGAGAAGG None C¹,sequence of deleted segments (del) is shown in FIG. 2

Cancer Related Sequences

The sequences of the following table (Table 3) may also be provided withthymine residues substituting the uracil residues (e.g. in DNA form).Each sequence of the following table may be an embodiment of thepolynucleic acid polymer of the invention. Each gene or ORF referred toin the table below (Table 3) under “name of compound”, may comprise thegene target for correction of intron retention.

TABLE 3 Name of Corresponding compoundnucleotide sequence of antisense oligoribonucleotide ABCD4-1SEQ ID NO: 47. UAGAGAGGUGUGGGAAGGGAAGCAGA ABCD4-2SEQ ID NO: 48. AAUUCCUUCAUCAUGGCACAUUUAUCCUUGCAGACAGG ABCD4-3SEQ ID NO: 49. CCUGAGGAUACUCACAGAAAGGCAACAG ABCF3-1SEQ ID NO: 50. UUUCCCCAACACACUCCAGCA ACADVL-1SEQ ID NO: 51. GGGCCGCUGCCCACCGUC ALKBH6-1SEQ ID NO: 52. CAGCACAGCUCAGAAGUCUGAG ALKBH6-2SEQ ID NO: 53. CCAAGCCAGGGACAGGGAGGUGAAUGCC AP1G2-1SEQ ID NO: 54. CUUCUGCCCAGCUCUCUGACUG APEX1-1SEQ ID NO: 55. UCUUCACAAACCCCUGCAAAAAUGAG ARFRP1-1SEQ ID NO: 56. CCCAAAGCCCCCGCAGGUGCAGCC ATHL1-1SEQ ID NO: 57. CCCCUCCCCACGCUCUGGAAA ATHL1-2SEQ ID NO: 58. GCAGCACCGGGAGGCUCAGACAAC ATHL1-3SEQ ID NO: 59. GAGCCUCAUCAAAGAAACGG ATP13A1-1SEQ ID NO: 60. GCUCCCACUGGGACUGAGCG ATP1A3-1SEQ ID NO: 61. AGAUGGGAAGAGAGAGAAGAG ATP1A3-2SEQ ID NO: 62. AGAGACAAGGAAACCACACAGACAGAGACC ATP1A3-3SEQ ID NO: 63. GCCGCCCAGCAGAGAGAGG ATP5D-1SEQ ID NO: 64. AGCUGGCUGGGCCCACCUGGCAU ATP5D-2SEQ ID NO: 65. GGGCCCAGGCAGAAGCCU ATP5D-3SEQ ID NO: 66. UCCCCAGAGCUUUCAACACAG ATP5D-4SEQ ID NO: 67. GCAGCCACAGCUCAAAGCUGAGGA BAX-1SEQ ID NO: 68. GAUCAGACACGUAAGGAAAACGCAUUAUA BAX-2SEQ ID NO: 69. GCAGAAGGCACUAAUCAAGUCAAGGUCACA BAX-3SEQ ID NO: 70. CGGGCAUUAAAGAGCUGGACUCAG BDH2-1SEQ ID NO: 71. ACCAAUUUUGAAAAAAGCAG BDH2-2SEQ ID NO: 72. CCACAUUUUAAUUUAAUUUUAC BDH2-3SEQ ID NO: 73. CCAUUAGAAAGAAUAAAAG BDH2-4SEQ ID NO: 74. UAUUUUAAAUUAAUUAAAUGUUAAAUGG BDH2-5SEQ ID NO: 75. AUUUCAUUUUAAACUCACAGAU BDH2-6SEQ ID NO: 76. AUCCUUGCAAAGAGAAGAAAUG BDH2-7SEQ ID NO: 77. UCCUUCAACUUGACUUCUUGCUGAUGGCUCAGAUCAACU BRD2-1SEQ ID NO: 78. UAUUUUAUAAAAGUAAAAUGCCAAGAACCAAAGACU BRD2-2SEQ ID NO: 79. UUCAAACUCCAAGAAAUACAAAUUCUCAAAACAC BRD2-3SEQ ID NO: 80. UUUUCUCAAGACAAAGAAACCC C16orf59-1SEQ ID NO: 81. GGGUGGAGCAGUCAAGCC C16orf59-2SEQ ID NO: 82. ACUUCCCAACCCACACACACAGAC C1orf124-1SEQ ID NO: 83. GUCACAUAAAAAUCAGAAGAAU C1orf124-2SEQ ID NO: 84. CAAAUAUUAUCAGAGAUUGAA C1orf124-3SEQ ID NO: 85. CUUGAAUUAUUGUUUUUAUUUUGACAAUC C1orf124-4SEQ ID NO: 86. ACUCAAUAAUUAAAGAUUUGGGAAAUAU C1orf124-5SEQ ID NO: 87. AGGCAACAUUUACCUUGAAAAU C1orf124-6SEQ ID NO: 88. GAGGGCAAUCUUCAGAAUUCAG C1orf63-1SEQ ID NO: 89. UAAUCAGAUUUGACAGUUGGCUUUCUGAAAGUUUU C1orf63-10SEQ ID NO: 90. ACAUUUCUGGAGAAUUAUAAUAAACUUAU C1orf63-11SEQ ID NO: 91. CAAUUACACAGAUUCAUUUAGAUA C1orf63-2SEQ ID NO: 92. UCAGAUUUGCUACUUUGAAUUUAGCACAUUAU C1orf63-3SEQ ID NO: 93. AAAUAAAGCUCAUUAAUCUCCCAUUUUCAUG C1orf63-4SEQ ID NO: 94. UGAAAAUGAAAAAAAUAAAUGU C1orf63-5SEQ ID NO: 95. GCUACAAACACUCUGUAAAUAGCUUAGAAAAACU C1orf63-6SEQ ID NO: 96. CAUGAUUUCUAUAAGACAGAAAUAGAGCAGAUAA C1orf63-7SEQ ID NO: 97. CAAUUACCAACAGAUUUUCUUCAUCAAUG C1orf63-8SEQ ID NO: 98. ACAUAAACUUCAAAUUAAACCU C1orf63-9SEQ ID NO: 99. GUACCUUUGCUUAGUUUAAAAAUUG C2orf49-1SEQ ID NO: 100. GGAAUUUGAUAAUUUUCUAAAGG C8orf82-1SEQ ID NO: 101. CGGAAGGGAGAAAGAAGGG C8orf82-2SEQ ID NO: 102. CCUGGCCUCACUCAGCG CAPRIN2-1SEQ ID NO: 103. UAAAGAAAUAAUGCUUACUGGU CAPRIN2-3SEQ ID NO: 104. UGUGGUAAUCAAAGCAAAUAGA CAPRIN2-4SEQ ID NO: 105. AUGAUUUAGAACAGCAUGAAAAAUCAAAAUA CAPRIN2-5SEQ ID NO: 106. CUUAAAUUUAAAUUAAGAAAUGAG CAPRIN2-6SEQ ID NO: 107. UAAAAGAAAAUGGAUUCUAAUUAAUAU CASKIN2-1SEQ ID NO: 108. GCAAAGCCACAGCUGAGGGUGACAGCACG CASKIN2-2SEQ ID NO: 109. CCAGCCAGAGGAGAAAAGGCA CDCA7-1SEQ ID NO: 110. CACACAAAUAAAGAAAUUAGAUUU CDCA7-3SEQ ID NO: 111. UUUUCUUCUUUUAUUUUCAUUCUCCAAUUUUAAA CDCA7-4SEQ ID NO: 112. AAGCCAGGAAAAAGAAAUCUUUUCUAUCA CDCA7-5SEQ ID NO: 113. AGAAACACAUUCAGUUUCUAC CDCA7-6SEQ ID NO: 114. UCUAAAAAAAAAAAUUUUCUC CDCA7-7SEQ ID NO: 115. UGCAUAAUGCAUGGCAAAAUGAGC CEP164-1SEQ ID NO: 116. GCUAGAGAAGCUAUGACUCUGAGGUCAAGGAC CEP170-1SEQ ID NO: 117. CUUCAUCAAAGAAUGCAAUCA CEP170-2SEQ ID NO: 118. ACUUUGAGUAAAAGAAU CEP170-3SEQ ID NO: 119. CUUUGCUUUCUCAAGUUUUGUAUGU CLCN7-1SEQ ID NO: 120. CCAGCAGAGGCAGGCAGAGAAGGAAG CLCN7-2SEQ ID NO: 121. CUGAAAUGAGAAACAGAAGACACAUAAGAGAACCC CLCN7-3SEQ ID NO: 122. GCCGCGUACAUACACACAGAACAACC CPNE1-1SEQ ID NO: 123. UGAGCAUCCCUUGGGCCUCAACCCUACUCACAUCAGGGAAAGGUGAAAGGGUAAACU CPNE1-2 SEQ ID NO: 124. AGGCUUAGAGGAAAAGGUGAGCAUCPNE1-3 SEQ ID NO: 125. UAUUUCAUGCUCAAGAACCCAACCA CPNE1-IVSB-SEQ ID NO: 126. CACAUCAGGGAAAGGUGAAAGGGUAAA 1 CPSF3L-1SEQ ID NO: 127. CCCACGCCGCCCGCCCG CPSF3L-1SEQ ID NO: 128. UCUGAGGCCCAGGGUCCAGCUGUGGAUG CPSF3L-2SEQ ID NO: 129. CAGCCAUCCAAGCACAACCACUGCU CPSF3L-4SEQ ID NO: 130. CUCACUGACAGAUGUGAGCUGGAAGCUGA CPSF3L-5SEQ ID NO: 131. GGGUUCUAUGUGCAGACUCAG DCXR-1SEQ ID NO: 132. CAUCACUCACGAGAAUUCC DENND4B-1SEQ ID NO: 133. ACAGACCAGGGAUCACCCAGA DFFA-1SEQ ID NO: 134. UUUAGAUUAAUGAGAUUUUUGC DFFA-2SEQ ID NO: 135. UGCAUUUUUCUUUAAAGCUAUUUUG DFFA-3SEQ ID NO: 136. AAGACCCAGAAGCCAUCUCAGAAGAUUG DFFA-4SEQ ID NO: 137. AUGACAGGGACAAGGACAAUGAAUCAGAAGUAG DFFA-5SEQ ID NO: 138. UUUUCUUACAACACCAACAGGAAGAAGU DFFA-6SEQ ID NO: 139. GUUUAUGUUACCUCUUUACACUGAAAUG DIS3L2-1SEQ ID NO: 140. GGGACACAGAUGAAGGAAUGAG DIS3L2-2SEQ ID NO: 141. CAAGGAAGGGAAGGUGGUGCCAGAAAGCAGG DIS3L2-3SEQ ID NO: 142. AGGCUUAUGAAACACAACC DNAJB12-1SEQ ID NO: 143. AGGGCCAAAGCUGCCAGGAGU DNAJB12-2SEQ ID NO: 144. CUCCCUUUCUCCCCCUCCCUCCUCUGCUCA DNAJB12-3SEQ ID NO: 145. CUGGAGCCAGGGAGCAGAGCG DNAJB12-4SEQ ID NO: 146. CUCAGCAACAGUUUCAAGUUCCCAC DNAJB12-5SEQ ID NO: 147. CCGCCACCAAGACUGCCAGCUCCCACCCACCUC DNAJB12-6SEQ ID NO: 148. AGUGCCUCAGAUCCCACCAGAGG DNAJB12-7SEQ ID NO: 149. GCCUGCUACCAGCAACUCUCAUUUCC DNAJB12-8SEQ ID NO: 150. CACAGAGAAGAACCUUCACUGCUUCUGC DNAJB12-9SEQ ID NO: 151. GAGGACACAGGCAAAGGAGGG DNAJB12-SEQ ID NO: 152. CCAAAGCUGCCAGGAGUUGCA IVSB-1 DNAJB12-SEQ ID NO: 153. GCUGGAGGUCAGGCUGGG IVSB-2 DNAJB12-SEQ ID NO: 154. CCCUCAGCAACAGUUUCAAGUUC IVSB-3 DNAJB12-SEQ ID NO: 155. AAUAGUCUGCUGUGCUGGAGAAAGGG IVSB-4 DNAJB12-SEQ ID NO: 156. UUCCUCCUAGCUGGAGGGAUGGAGAAAG IVSB-5 DNAJB12-SEQ ID NO: 157. AGAGAGUGCCUCAGAUCCCACCAGA IVSB-6 DNAJB12-SEQ ID NO: 158. AGAAGGAGGGAGCCUGCUACCAGCAACUCUCAUUUC IVSB-7 DNAJB12-SEQ ID NO: 159. CACACAGAGAAGAACCUUCAC IVSB-8 DNAJB12-SEQ ID NO: 160. CAGCACAGAGGCAGGCACAAAUG IVSB-9 DPF1-1SEQ ID NO: 161. UCUGGAACGGGAGGGAGAGGG DPF1-2SEQ ID NO: 162. CUCAGCCAGAGACCUGAGCAGC DRG2-1SEQ ID NO: 163. CAAUUUCAACGAUCAGUAACAGAGC DRG2-2SEQ ID NO: 164. UUCUGGAAAGCGGGAUAAUGGAC DRG2-3SEQ ID NO: 165. CAUCAUAAAAGGAGUAACAGGAUAAUA DRG2-4SEQ ID NO: 166. CUUAUUUCAGAAGAAAAUCCGA DRG2-5SEQ ID NO: 167. CAAGCUUGGCAUUUUUCUUUAAUCCA DSN1-1SEQ ID NO: 168. GUGGAAACAUAAAGAAAGCAUC DSN1-2SEQ ID NO: 169. UGCAAAAAAGUGGAAAAAGUAAAUGUA EML3-1SEQ ID NO: 170. AUCUUCAGGUUUCUGGACUCUCACCCA EWSR1-1SEQ ID NO: 171. AAACAAAAUUAGGUAAAAGGAG EWSR1-10SEQ ID NO: 172. CUUUAAACACAAAAGUUUACA EWSR1-2SEQ ID NO: 173. GGAAAUGCAGAAAUUAAUUUCUUAUG EWSR1-3SEQ ID NO: 174. AUUUCAAGACAACCAUUCAAAGGCAGUUAGUUAACAA EWSR1-4SEQ ID NO: 175. CUAAACAAAGUUUUCUAAACCAGAUU EWSR1-5SEQ ID NO: 176. GGACAGAACACACACAGAAC EWSR1-6SEQ ID NO: 177. AGUUAAAAAUCAACUUUAAUUUUGAAG EWSR1-7SEQ ID NO: 178. UUUUCCAAAUCAGAAGAUUG EWSR1-8SEQ ID NO: 179. UAUUUUAAAACAUCCAAAAAGAAGU EWSR1-9SEQ ID NO: 180. GACAAAGCAUGUUAAAAAGUUUCCA FGFR4-1SEQ ID NO: 181. AUCAGAUGAGCAGCAGCGG FTSJ1-1SEQ ID NO: 182. GGGUCAAGGCAGGCUGAGAG FTSJ1-2SEQ ID NO: 183. CCAGAAACCAUGAGAUUUGGGUCAGAAAAAGGCA FTSJ1-IVSB-1SEQ ID NO: 184. CAGUCGGCGUCCCAGAGAUCC GBAP1-1SEQ ID NO: 185. CAUUUAAGUAGCAAAUUCUGGGC GBAP1-2SEQ ID NO: 186. CUCAUCUUCUUCAGAGAAGU GBAP1-3SEQ ID NO: 187. CCAAAGAAUUGGCAAAGAAAAG GBAP1-4SEQ ID NO: 188. AUUUCACUGGCAUUAAGACAG GBAP1-5SEQ ID NO: 189. GUCCGUAGCAGUUAGCAGAUGA GBAP1-6SEQ ID NO: 190. GUCUGAGUCAGGGCCAAAAGGAA GMPPA-1SEQ ID NO: 191. GGGAAACAGCAUGAAGAUAAGCAGG GMPPA-2SEQ ID NO: 192. AUGAGAAACUAGAUUAGGG GMPPA-3SEQ ID NO: 193. GAAAAGCAAUAAAGAAAUGAGCAACA GMPPA-4SEQ ID NO: 194. AAGUCCAGAAACCAGUUUCAGUC GMPR2-1SEQ ID NO: 195. GAGCUGGGAAAGGGUUGUGAGAGAC GMPR2-2SEQ ID NO: 196. GGUCCCUGAAGCCUGUCACC GMPR2-3SEQ ID NO: 197. CGCUUAAGUUGUGGAAGGUCG GNPTG-1SEQ ID NO: 198. AGCACUACAGGGCCUCCAGCAGGGC GORASP1-2SEQ ID NO: 199. ACAAAACCAGACACUUCUCAUGGACAGCA GPATCH4-1SEQ ID NO: 200. AUCUGAAGACAUCUCUUCCCACAUU GPATCH4-2SEQ ID NO: 201. CCAGUCAAGCAUUAGAUUUAGC GPATCH4-3SEQ ID NO: 202. UCCUCCUUCUAAAACAUU HGS-1SEQ ID NO: 203. AGGAUGCACCCCAUGCU HMG20B-1SEQ ID NO: 204. CGGAGCCACAAGCAAUUCAAAUCCAGC HMG20B-2SEQ ID NO: 205. GUCAGCAGUCGGGACACGGUGGGUUAGA IFFO1-1SEQ ID NO: 206. UGGUUAAAGAAACUGGAGAAAGAAAAGCAAAAGGAUAAAG GAA IFFO1-2SEQ ID NO: 207. CAAGUCAGGGAGAGAGAGAGAGAGG ISYNA1-1SEQ ID NO: 208. AGCCGCCCCGCUCUCCCCAGC KRl1-2SEQ ID NO: 209. GAGGAUGAAAGAGGAAAGG KRl1-3SEQ ID NO: 210. CUGAGGGCACAAGAGAGACAG KRl1-4SEQ ID NO: 211. GGGAAGACAAAGACUUGACAAGG KRlI-5SEQ ID NO: 212. AGGUCAAACAGGUGGUCAAACAGCAGGA LOC148413-SEQ ID NO: 213. UAAGGACUGAAGACACGACG 1 LOC148413-SEQ ID NO: 214. GAGUGUUGAAGGCAAGACUUUGCAG 2 LZTR1-1SEQ ID NO: 215. CCCACUCAGUGGGAGCUGCAGCCAU MAN2C1-1SEQ ID NO: 216. GGAAGACCCAUUUCUCCAUGCC MAP4K2-ISEQ ID NO: 217. CCCAGAGCUCUGAGGGUGCCCUGGGC MCOLN1-2SEQ ID NO: 218. GUGCUCACCCAGCAGGCA MCOLN1-3SEQ ID NO: 219. GCCACGUGCUGACUCUGCAGCUGGCAGG MDP1-1SEQ ID NO: 220. UCGCCCCCAGUCUUCCCU MIB2-1SEQ ID NO: 221. GGCAGCACAGCAAGAGG MITD1-1SEQ ID NO: 222. CAAAAACAGUGCUACACAUUUACUCA MOK-1SEQ ID NO: 223. UCAGAAAGCCUGUGACAAAUCUU MOK-2SEQ ID NO: 224. AAGAAGAGUCCAAAAUGGUU MOK-3SEQ ID NO: 225. UGAGAAGAAUGAAAUAAAAUUUAACAAAA MOK-4SEQ ID NO: 226. UGUUAUGCUAAAAAUGUAAGAAAAC MOV10-4SEQ ID NO: 227. AUCAGAAUUUCCAAGAGAGAGGCC MOV10-5SEQ ID NO: 228. UAAGGAAAGAAAACAGCAUUGCAAAGAACACG MRPL35-1SEQ ID NO: 229. AGUUUUAAAACUUUUCUAAGUUUAAUGU MRPL35-5SEQ ID NO: 230. AAUGAAAACAUGAAAUCUGA MRPL35-6SEQ ID NO: 231. GAAAAUUUGUGGGAAAAGUUUAUCCUUAC MRPL35-7SEQ ID NO: 232. UCUGAAACAGUAAUUCAUGCAUAAUUCU MRPL35-8SEQ ID NO: 233. UGCAGAACUUCAAUUUCAUAAUUUU MTMR11-1SEQ ID NO: 234. AAACAAAUCAAGACCAAACUUCAGAGAGU MTMR11-2SEQ ID NO: 235. CCUGAAAAUGAGAAUAAAUCUCC MTMR11-3SEQ ID NO: 236. GACAAAUCAUGAGAUUCUCACC MUS81-1SEQ ID NO: 237. UCCCUGCCACUCCCUCCA MUS81-2SEQ ID NO: 238. CUGCAGGAAGAGAGGCAGCGA NAPEPLD-1SEQ ID NO: 239. GCCUUUUUCAUUAAAAG NAPEPLD-2SEQ ID NO: 240. UUUCAUUUGUUUUUAAACUUAGAU NAPEPLD-3SEQ ID NO: 241. UAUUCAUGAAUUUCUAA NAPEPLD-4SEQ ID NO: 242. UUUCCAAAUGUAAAAUAAUCACA NAPEPLD-5SEQ ID NO: 243. CACAAAACAUAAAACAUAAAC NAPEPLD-6SEQ ID NO: 244. UACUAGGAAGCAAGUUAUUA NAPEPLD-7SEQ ID NO: 245. AAUUCAUUAUUUAAAUGAC NAPEPLD-8SEQ ID NO: 246. AUGAAAUUUAAAAUCCACAUUAGC NBEAL2-1SEQ ID NO: 247. ACAUUCUGAUUAGGGAGG NDRG4-1SEQ ID NO: 248. GAAGGCAACAGAGGUGAGUGUGA NDRG4-2SEQ ID NO: 249. CCAGAGGGCAGGCAAGGCAGAAGUG NDUFB10-1SEQ ID NO: 250. GGAAGAUUUGCAAUGGUUCUG NFATC4-1SEQ ID NO: 251. ACACACAGACAAAAGAGUUGCAAGAGACAGAGAC NFATC4-2SEQ ID NO: 252. GGCAAACUAGAAUAGAAAGA NFATC4-3SEQ ID NO: 253. CCAGAGCAGAGAGAGGGUUAAACAGG NFATC4-4SEQ ID NO: 254. UCAGCAGUAGACACACAAAUAAACCAG NFKBIB-1SEQ ID NO: 255. GUCGGUGCCUAAUUAUCUUCUUGGG NFKBIB-2SEQ ID NO: 256. AGUUUUUCAGCCACUUCU NFKBIB-3SEQ ID NO: 257. UCUUGCUGCCUAAAAUCAC NFKBIB-4SEQ ID NO: 258. UGCCUUUACCCAAAUUCCUC NFKBIB-5SEQ ID NO: 259. UUCAAGGUCAUUUCUACAGACCAAUUUCU NIT1-1SEQ ID NO: 260. GGACACUGUCCAACAAAGAUUCUAC NIT1-2SEQ ID NO: 261. CUGGCAACCCAGGGACAC NKTR-1SEQ ID NO: 262. AAUAAAAUUGAGUUUAUAGAAUUA NKTR-2SEQ ID NO: 263. AUUUGCCAGAUUUCAAUUUAAAGUUUAAAAAG NKTR-3SEQ ID NO: 264. AAACUGAAAACACACAAAUCUUUGAAAUGAAAUGC NKTR-6SEQ ID NO: 265. CUUUUUUAUUUUAAGAGUUCCA NKTR-7SEQ ID NO: 266. AUGAUUUUCACAAAGAGAACAAUA NKTR-8SEQ ID NO: 267. AUUUCAUAAUAAAAGCACAUAAAAUUAGU NPRL2-1SEQ ID NO: 268. CCUGCCACCCACCGCUCACCC NPRL2-2SEQ ID NO: 269. CCUUCCUCCUCCUGGGACAA NSUN5P1-1SEQ ID NO: 270. AUUAAAGUGUCAGAACUAAGACCAAAACAGAUG NSUN5P1-2SEQ ID NO: 271. CCUGAAAUCCUUGCCUCACAGAGGAGAACU NSUN5P1-3SEQ ID NO: 272. GCCUCAGUCCUGAAAUCCU NUDT22-1SEQ ID NO: 273. GGCAGUAAAACGUGCCAUCUUC NUDT22-2SEQ ID NO: 274. UGUCGCAGACCUCCUGAGGG PAN2-1SEQ ID NO: 275. UCUUCCUUUUCCCUCUGCUAAGUUU PAN2-2SEQ ID NO: 276. GUGACUAUGGAAAAUCCCCUAACAG PDDC1-1SEQ ID NO: 277. GUGCAGCUCUGAUGUGGCAGG PDLIM4-1SEQ ID NO: 278. UGCAGGGAGUGGGAAGGCAGAU PDLIM4-2SEQ ID NO: 279. GGGCCGCAGAGACCGAAGAGGGCAGGUG PDLIM4-3SEQ ID NO: 280. GAAGCCAGGGCGUAGCAAGGUUGUAGCAA PDLIM4-4SEQ ID NO: 281. GGGCAACCUGGGCACUGCA PHF1-1SEQ ID NO: 282. UUUUUCCUUCAUUUCCUGGGAU PHF1-2SEQ ID NO: 283. GUCCCAAACCCUAAACUUACCUC PIK3CD-1SEQ ID NO: 284. CUGGGAUUCCCACAGAACGG PIK3CD-2SEQ ID NO: 285. GCUGGAAACGUCCCCAGUGGCCUUCC PITPNM1-1SEQ ID NO: 286. GGCGGAGCCCUCCCGCAGAGGC PPIL2-1SEQ ID NO: 287. GCAGCAGGCAAGCAAUUUAUUG PPIL2-2SEQ ID NO: 288. GCCCUUGGCAACAGGUUAAGGGA PPIL2-3SEQ ID NO: 289. CAGGUCCUGGAAACAGGGCCAA PPIL2-5SEQ ID NO: 290. GGGUAAGAAAACCAGACAUA PPIL2-7SEQ ID NO: 291. GCACAUUUAACAGAAAAAUG PPIL2-8SEQ ID NO: 292. UGAAGACGAAGAAAAAGCCAGCCAGG PPP1R35-1SEQ ID NO: 293. CGCACGCGGCCGGCCGCCCGC PPP4C-1SEQ ID NO: 294. CCACCCCCAAAAGCAGAAU PPP4C-2SEQ ID NO: 295. CUGCCCCUCCCAGAAUGCUG PPP4C-3SEQ ID NO: 296. UCUUUCACCUACCAGACACAGAC PPP4C-4SEQ ID NO: 297. CCUCCAGAGAAUGUAAAGCUGA PQLC2-1SEQ ID NO: 298. GGAGAGGGCUGGAAGGAUGUGGCA PQLC2-2SEQ ID NO: 299. AAAAACGAAGCCAUCAGAUGCCAAG PRPF39-1SEQ ID NO: 300. GUGACAAAUGCAAAUAAAUAC PRPF39-2SEQ ID NO: 301. CUGCCAACAAAGAGAGAAAAUAUUAGCU PRPF39-3SEQ ID NO: 302. UGUUUGGAAAAUGAGAAAUAAAUGU PRPF39-4SEQ ID NO: 303. UAGCAAAUGUGACUAGCAAACCAAC PRPF39-5SEQ ID NO: 304. CUAAUUACUGGAAUUUUGUUUAAAUAAUC PSME2-1SEQ ID NO: 305. UUGUUAGCUAGAGAGGGUGGGCAAAGGG PSME2-2SEQ ID NO: 306. CCUAAUCCACUAUUUGAAAC PSME2-3SEQ ID NO: 307. CAUGCCUCACGCCAUCCUAAUG PTPMT1-1SEQ ID NO: 308. GACAGGGACGGAGCGGCGG QARS-1SEQ ID NO: 309. ACCUCCCUCACCCCAAACC RAD52-1SEQ ID NO: 310. GGCCGCAGAGGAAAGGAGG RAD52-2SEQ ID NO: 311. GCAGCCCCGUGACACAGGAG RHOT2-1SEQ ID NO: 312. CACAGGCCGCGCCGCCCC RHOT2-2SEQ ID NO: 313. CCAUGCUGGGCCAGAUCUGCCAGG RMND5B-1SEQ ID NO: 314. CUGAGAGGUCGAAGCAGAAUGC RMND5B-2SEQ ID NO: 315. GUGAAAUGAAGACCACAGUCAAGCCC RMND5B-3SEQ ID NO: 316. GAGACAGGGCUGCAGGCAAGUCAAGUA RNF123-1SEQ ID NO: 317. ACACACACAACCAAACACGCACAACAC RNF123-2SEQ ID NO: 318. GGCAGCAGGAGCAGAAACCAG RNF123-3SEQ ID NO: 319. CACAACAGUCAGCAGGUCAGACUG RPL10A-1SEQ ID NO: 320. GAAGGGUCUGGGACCGCAGCA RPP21-1SEQ ID NO: 321. UACAGUGAGAAAGGCGCU RPP21-2SEQ ID NO: 322. AGGAACUUAAUCCAAACCCGAAGAAGGAAGAC RPP21-3SEQ ID NO: 323. CCUCUUAAAAGUUAUUAUUUAUU RPP21-5SEQ ID NO: 324. AAUUUCAAUGAGAAUAAUGAAU RPP21-7SEQ ID NO: 325. UCUUUAAGAUAAAGUUCAAAC RPP21-8SEQ ID NO: 326. CAAUUUGAAUGCACAUUUGAU RPS6KB2-1SEQ ID NO: 327. CGACAGACGUGGCCAAGGCA RPS6KB2-2SEQ ID NO: 328. AGACACAGCAACCGAAGCCAACACU RPS6KB2-3SEQ ID NO: 329. ACACAGGCCGCGGGCUCCACAAAC RUSC1-1SEQ ID NO: 330. GAGCUCCAUUACUCUCCUCAU RUSC1-2SEQ ID NO: 331. CACCUCCCGCCAACCAUUCC SCRN2-1SEQ ID NO: 332. UUCCUUCAUAUUUCCAGAGUC SCRN2-2SEQ ID NO: 333. UCCCCAGCUCUGAAAUCUCU SCRN2-3SEQ ID NO: 334. CUCACACAAGCAGGAGAAAGGAGAU SCYL1-1SEQ ID NO: 335. CUAGUCUUCAGCCCACCCAG SFR1-1SEQ ID NO: 336. ACAAUACUUAGAAACACAUAAUGG SFR1-2SEQ ID NO: 337. CGUAGAAUUUAAACCACC SFR1-3SEQ ID NO: 338. CACAUUAUGUUAAUUAACAAC SFR1-4SEQ ID NO: 339. AGAAGAAAAACAAAAUUAUUUAAUAAAAU SFR1-5SEQ ID NO: 340. UAACUGAAAUGAAUUCAUUCAAGAGGAAAAUAUGGAA SFR1-6SEQ ID NO: 341. UCAGAAUUACAGAGUAAGGAAAAGACCU SFR1-7SEQ ID NO: 342. GGCAUCACAAAAUGACUUUAAUUUCUGGA SGSM3-1SEQ ID NO: 343. CUAACCCCAGAGAGGUCUCUA SIRT7-1SEQ ID NO: 344. UGGAGACCCUGGGUCCCUGCAG SLC25A3-1SEQ ID NO: 345. CCACAGGAGCUCUGGGCU SLC25A3-2SEQ ID NO: 346. UGGGCCCACCGCCAAAGCAGCG SLC25A3-3SEQ ID NO: 347. UCCACGCCCUUGAAGAGGUCACGGCGG SLC30A7-1SEQ ID NO: 348. AUUUCUCUCUUUUAAAAAGCUG SLC30A7-2SEQ ID NO: 349. GCACAAAAGAAAAGACCAAAAGU SLC30A7-3SEQ ID NO: 350. CAGAAGUCAAAAAGAUUUGGAGGAAAG SLC30A7-4SEQ ID NO: 351. AAACCUCAGAAGUCAAAAAGAU SLC37A4-1SEQ ID NO: 352. UAUGACAAUCCAAACAGGC SLC37A4-2SEQ ID NO: 353. UAAGAAAGGGCGCUCCCACAUGCUCUUUAGG SLC37A4-3SEQ ID NO: 354. UCCUAAAAUAUCUUGACAAGCAAU SLC37A4-4SEQ ID NO: 355. AAGCUCACAUUACAGGGAAGAGGGA STK19-1SEQ ID NO: 356. UCAUUUUAUUAACAAGAAGAGUC STK19-2SEQ ID NO: 357. ACCAAGAACUGAAUUCUAUUUCAGG STK19-3SEQ ID NO: 358. GAAACACGGGCAACCAUGCAAGAGAGACU STX10-1SEQ ID NO: 359. CCCACCAGGACUGACCCCUCCC TCF25-1SEQ ID NO: 360. CCCUCCUGCUGCUGGAAGCAGGUCC TCF25-2SEQ ID NO: 361. GUCACAGAAAUGUGAAAAUGCACC TCF25-3SEQ ID NO: 362. UUCUUUAGGAAGCAGGACUGA TOMM40-1SEQ ID NO: 363. GACUCAGCCCCAGCAAAUCCGC TOMM40-2SEQ ID NO: 364. GCACCCGGCUCCGGCCCC TP53I3-1SEQ ID NO: 365. GCAAAUCACACUCCCUCUGAGUUGGAAGC TP53I3-2SEQ ID NO: 366. CCGCCUCCAGACCGAUCCCACCCGGAACACAGAUGGG TRIM41-1SEQ ID NO: 367. AUACCGAAGAGAAGCAGGGAC TRIM41-3SEQ ID NO: 368. CCCAGAGGGAAAAGCAAAAGCUGAGG TRPT1-1SEQ ID NO: 369. GCAGACAGGCUCACGUUUCUCU TRPT1-2SEQ ID NO: 370. CCCAGACAAGAACUCUCCUCAG TSTA3-1SEQ ID NO: 371. GCUGGGCCUCAGCAGGA TSTA3-3SEQ ID NO: 372. CUUACUGAGGCUGGCACGAAGACC TTC14-1SEQ ID NO: 373. CCUUAAGUUUAAAAAUACUGA TTC14-10SEQ ID NO: 374. AAAUGUUUCUAAAUUAUUCAUAAAGAUG TTC14-2SEQ ID NO: 375. AAUACUUUCAUAUUUUUAUUUACUUUACCUCC TTC14-3SEQ ID NO: 376. UCUUUAAUAAGAAAAUACAUGGAACACA TTC14-4SEQ ID NO: 377. UAUUCUAUAUUUUAAUUCUAAGAUACUCU TTC14-7SEQ ID NO: 378. UGAAAGACAGACUUUUUUCAACACUACCUUAAAAACUUAA GAC TTC14-8SEQ ID NO: 379. AAGAUCUAAUUUUACUAUUAAGCAC TTC14-9SEQ ID NO: 380. UAUUUGUUUCCUUUAAAGAUUUUAUAAAAGCU TUBGCP6-1SEQ ID NO: 381. CCUGCCAACAGCAACUGC TUBGCP6-2SEQ ID NO: 382. ACGUGCUGGGAACCAGCCAGC TUBGCP6-3SEQ ID NO: 383. UCCGCCCCCAUCCACAGGAGAUG U2AF1L4-1SEQ ID NO: 384. GGCUUAGGGUUAGGCUCAUCUGAGGAU U2AF1L4-2SEQ ID NO: 385. CUGAAAUAACUAGAGUUCUAAGACACGA UCK1-1SEQ ID NO: 386. CAGGACCUGCCGCCAGCCUCGGCCAGGCAGGCACGG UNC45A-1SEQ ID NO: 387. CCACAGAAGCCCUACAGCUCC UNC45A-2SEQ ID NO: 388. CGGUGCAGCGGUCCCAGAGUCC VAMP1-1SEQ ID NO: 389. AGGCUUGUCCAUCAAAGAAAUC VAMP1-10SEQ ID NO: 390. AGGGCGAAAGGAAAGGAAGGAUG VAMP1-2SEQ ID NO: 391. AGCCCCACUUCCUCAGAACAGG VAMP1-3SEQ ID NO: 392. GGAAAAGAGAAAGAGACAGGAGAAAACAAGAGGGU VAMP1-4SEQ ID NO: 393. AACUUGAGAGUACAGAAAAAGCAGG VAMP1-5SEQ ID NO: 394. CCAGUGGCCAGGUUUUCUAGA VAMP1-6SEQ ID NO: 395. ACGAACAGAUUAGAAAUAACU VAMP1-7SEQ ID NO: 396. CUGUAGAAAAUGUAAAGAAGAGAAAGC VAMP1-8SEQ ID NO: 397. UAGAAUUCAGACAGGAAAGGG VAMP1-9SEQ ID NO: 398. CAAACCAUGCAAAGAGGAGGAAGAGAAA VARS-1SEQ ID NO: 399. CCUCCAGACCCUCAAAGC VPS28-1SEQ ID NO: 400. CCGCCUGGCUGGGAGGG WDR24-1SEQ ID NO: 401. AGCAGCCCCAGCCCCUGG WDR90-1SEQ ID NO: 402. CCCCACCCACAGUGCCAG WRAP53-1SEQ ID NO: 403. CUCAGGGAUCCGACGCAGAG YDJC-1SEQ ID NO: 404. UGUUUGAAUGCGGAAGUCAUCC YIPF3-1SEQ ID NO: 405. AUCCUCAGGCAGCUUUCAACC YIPF3-2SEQ ID NO: 406. UGAUCUCAGCCUCACCUAG ZCCHC18-1SEQ ID NO: 407. CACAGAUUUAUGAUAAUAAGAAACCAUUA ZCCHC18-2SEQ ID NO: 408. CUUCUAAUUCUAGAUGACAUAG ZCCHC18-3SEQ ID NO: 409. GCCGCUUCCGUUUAAUAAAAGCAUC ZCCHC18-4SEQ ID NO: 410. CUGGUAGAAAGAGACUGAGC ZCCHC8-1SEQ ID NO: 411. CUUAGUGGCAAGAUGCAUAAAAG ZCCHC8-2SEQ ID NO: 412. UGCAAAAUUUGGAAAUUGUUUUAA ZFAND1-1SEQ ID NO: 413. CACUUAAACAGAUAUACAAAGUGUGAA ZNF131-1SEQ ID NO: 414. UGACAGCUGAAGUUCCACAA ZNF131-2SEQ ID NO: 415. AUGGAACAAGUCCUUCACAU ZNF300-1SEQ ID NO: 416. UUCAGGAAAGACAACAAAUAUAAACA ZNF300-2SEQ ID NO: 417. UAUUUGACAUUUAAUUUAAUACA ZNF300-3SEQ ID NO: 418. UAAUUUUCUCUGAACUUCUAAAACAGU ZNF300-5SEQ ID NO: 419. CAACUAACAAAUAAUAGAAAAAUCCAA ZNF300-6SEQ ID NO: 420. UUAAUUUCAUUUAUAUUAUAAAUCAUGU ZNF300-7SEQ ID NO: 421. GACAGACAAGAAUGUUAAACAGAAAUA ZNF317-1SEQ ID NO: 422. GAAGCUCUGCAAGAAUUCCAGCAUGCAC ZNF317-2SEQ ID NO: 423. GGAAACAGAUGCUACAUAAAUC ZNF317-3SEQ ID NO: 424. GAGCAAGGGCCUGAGAUUUUGCAAGCAUG ZNF317-4SEQ ID NO: 425. CUUCAGAUGCAACCCUGACAAGGGACUAAU ZNF692-1SEQ ID NO: 426. GCCCCUGCCCUUUCUGUCUCA ZNF711-1SEQ ID NO: 427. GUUAAAACAUAGGUUAUAAAAGAAGAAC ZNF711-2SEQ ID NO: 428. UAGAAGAAAGCAAAACAACAAAACU ZNF711-3SEQ ID NO: 429. AGUAAACCAAAAAUAAUGG ZNF711-4SEQ ID NO: 430. UUUGAGAAAAAAAUGCAAUUGACAA ZNRD1-1SEQ ID NO: 431. AUUCUGUCCCAGGACCUAGGAGU ZWINT-1SEQ ID NO: 432. UGCAGAGCAGCUUGUCUUUCUUCUGAGAG ZWINT-2SEQ ID NO: 433. UACUCACGGCUCGUGUCUUCAGAAGCCAAGG ZWINT-3SEQ ID NO: 434. CCUUCCCCACUCAGGUCAGCUGCUA

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The particular methods, compositions, and kits described can vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

When values are provided, it can be understood that each value can beexpressed as “about” a particular value or range. “About” can alsoinclude an exact amount. For example, “about 5 μL” can mean “about 5 μL”or “5 μL.” Generally, the term “about” can include an amount that wouldbe expected to be within 10% of a recited value.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Unless defined otherwise, all technical and scientific terms used hereincan have the same meaning as is commonly understood by one of skill inthe art to which the claimed subject matter belongs. The descriptionsherein are exemplary and explanatory only and are not restrictive of anysubject matter claimed. In this application, the use of the singular caninclude the plural unless specifically stated otherwise. As used in thespecification and the appended claims, the singular forms “a,” “an” and“the” can include plural referents unless the context clearly dictatesotherwise. In this application, the use of “or” can mean “and/or” unlessstated otherwise. Furthermore, use of the term “including” as well asother forms, such as “include”, “includes,” and “included,” is notlimiting.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entirety tothe same extent as if each individual publication, patent, or patentapplication was specifically and individually indicated to beincorporated by reference.

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What is claimed is:
 1. A method of treating or preventing a disease orcondition in a subject comprising correction of intron retention in genetranscripts, the method comprising: administering to the subject apharmaceutical composition comprising: (i) a therapeutic agent thatinduces splicing out of an entire intron in a partially processed mRNAtranscript; and (ii) a pharmaceutically acceptable excipient and/or adelivery vehicle; wherein the disease or condition is treated orprevented in the subject by the administration of the therapeutic agent.2. The method of claim 1, wherein the disease or condition or apredisposition to the disease or condition is associated with adefective protein expression caused by the intron retention in the genetranscripts.
 3. The method of claim 1, wherein the disease or conditionis a hereditary disease, diabetes, or cancer.
 4. (canceled) 5.(canceled)
 6. The method of claim 1, wherein the therapeutic agent is apolynucleic acid polymer or a small molecule.
 7. The method of claim 6,wherein the therapeutic agent is a polynucleic acid polymer that is ananti-sense sequence that hybridizes to a retained intron of thepartially processed mRNA transcript.
 8. The method of claim 1, whereinthe pharmaceutical composition comprises: a polynucleic acid polymerthat hybridizes to a target sequence of a partially processed mRNAtranscript, wherein the partially processed mRNA transcript encodes aprotein and comprises an entire retained intron, wherein the polynucleicacid polymer induces splicing out of the entire retained intron from thepartially processed mRNA transcript; and a pharmaceutically acceptableexcipient and/or a delivery vehicle.
 9. The method of claim 8, whereinthe target sequence is a binding motif that forms a hairpin structure.10. The method of claim 8, wherein the target sequence is between two Gquadruplexes of a partially processed mRNA transcript.
 11. The method ofclaim 8, wherein the target sequence is within the entire retainedintron of the partially processed mRNA transcript.
 12. The method ofclaim 8, wherein the target sequence does not form a G quadruplex. 13.The method of claim 8, wherein the target sequence is an intronicsequence.
 14. The method of claim 13, wherein the intronic sequencecomprises an intronic splicing regulatory element comprising a first CCCmotif or a second CCC motif.
 15. The method of claim 8, wherein thepolynucleic acid polymer is from about 10 to about 50 nucleotides inlength, or is from about 10 to about 30 nucleotides in length. 16.(canceled)
 17. The method of claim 8, wherein the polynucleic acidpolymer comprises a sequence that is at least 60% complementary to thetarget sequence of the partially processed mRNA transcript.
 18. Themethod of claim 8, wherein the polynucleic acid polymer is modified at anucleoside moiety, at a phosphate moiety, at a 5′ terminus, at a 3′terminus, or a combination thereof.
 19. The method of claim 18, whereinthe polynucleic acid polymer comprises an artificial nucleotide.
 20. Themethod of claim 19, wherein the artificial nucleotide is selected fromthe group consisting of 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl(2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), T-O— dimethylaminoethyloxyethyl(2′-O-DMAEOE), 2′-O—N-methylacetamido (2′-O-NMA), a locked nucleic acid(LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a1′,5′-anhydrohexitol nucleic acid (HNA), a morpholino, amethylphosphonate nucleotide, a thiolphosphonate nucleotide, and a2′-fluoro N3-P5′-phosphoramidite.
 21. The method of claim 8, wherein thepolynucleic acid polymer comprises a sequence that is complementary to asequence with: (i) at least 80% sequence identity to at least 13contiguous bases of SEQ ID NO: 46; (ii) at least 10 contiguous bases ofSEQ ID NO: 46; (iii) at least 80% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, SEQ IDNO: 12, SEQ ID NO: 15, SEQID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQID NO: 27, SEQ ID NO: 30, SEQID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39,SEQ ID NO: 42, and SEQ ID NO: 45; (iv) at least 60% sequence identity toSEQ ID NO: 3; or (v) at least 80% sequence identity to at least 13contiguous bases of a sequence selected from the group consisting of SEQID NOs: 47-434.
 22. The method of claim 8, wherein the delivery vehiclecomprises a cell penetrating peptide or a peptide-based nanoparticle.23. The method of claim 1, wherein the subject is human. 24.-30.(canceled)